Functional diversification of cerato-platanins in Moniliophthora … · 2019. 12. 20. · Other cerato-platanins present diverse biological activities, such as the induction of systemic

Mario R. de O. Barsottini – MPMI

1

Functional diversification of cerato-platanins in 1

Moniliophthora perniciosa as seen by differential 2

expression and protein function specialization* 3

4

Mario R. de O. Barsottini a,b**

, Juliana F. de Oliveirab**

, Douglas Adamoskib**

, Paulo J. P. L. 5

Teixeiraa, Paula F. V. do Prado

a,b, Henrique O. Tiezzi

b, Mauricio L. Sforça

b, Alexandre Cassago

c, 6

Rodrigo V. Portugalc, Paulo S. L. de Oliveira

b, Ana C. de M. Zeri

b, Sandra M. G. Dias

b**, Gonçalo A. 7

G. Pereiraa,b and Andre L. B. Ambrosio

b** 8

9

aDepartamento de Genética e Evolução, UNICAMP, Campinas, SP, Brazil 13083-970 10 bLaboratório Nacional de Biociências, CNPEM, Campinas, SP, Brazil 13083-100 11

cLaboratório Nacional de Nanotecnologia, CNPEM, Campinas, SP, Brazil 13083-100 12

13

*Running title: Cerato-platanins function specialization in M. perniciosa 14

** These authors contributed equally to this work 15

16

To whom correspondence should be addressed: Andre L. B. Ambrosio or Sandra M. G. Dias, Laboratório 17

Nacional de Biociências, CNPEM, Rua Giuseppe Maximo Scolfaro 10000, Polo II Alta Tecnologia, 18

Campinas, SP, Brazil. CEP 13083-100. Tel.: (55) 19 3512 1115; Fax: (55) 19 3512 1004; E-mails: 19

[email protected]; [email protected]. 20

21

Nucleotide sequences were deposited at the GenBank under the accession numbers JX847578 (MpCP6), 22

JX422024 (MpCP7), EU250343 (MpCP8), JX422025 (MpCP9), JX847579 (MpCP10), JX422026 23

(MpCP11), JX422027 (MpCP12), JX422037 (SMpCP1), JX422028 (SMpCP2), JX422029 (SMpCP3), 24

JX422036 (SMpCP6), JX422030 (SMpCP7), JX422031 (SMpCP8), JX422032 (SMpCP9), JX422033 25

(SMpCP10), JX422034 (SMpCP11), JX422035 (SMpCP12), JX422041 (CcCP1), JX422043 (CcCP2), 26

JX422039 (CcCP3), JX422042 (CcCP4), JX422040 (CcCP5), JX422044 (CcCP6), JX422038 (MrCP1), 27

JX426104 (MrCP2), JX426107 (MrCP3), JX426108 (MrCP5), JX426106 (MrCP6), JX426113 (MrCP7), 28

JX426110 (MrCP8), JX426105 (MrCP9), JX426109 (MrCP10), JX426112 (MrCP11) and JX426111 29

(MrCP12). 30

31

The atomic coordinates and structure factors under PDB ID 3SUJ (MpCP1), 3SUK (MpCP2), 3SUL 32

(MpCP3) and 3SUM (MpCP5) are available at the Protein Data Bank. 33

34

35

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ABSTRACT 36

Cerato-platanins (CP) are small cysteine-rich fungal-secreted proteins involved in the various stages of 37

the host–fungus interaction process, acting as phytotoxins, elicitors and allergens. We identified 12 CP 38

genes (MpCP1 to 12) in Moniliophthora perniciosa’s genome, the causal agent of Witches’ Broom disease 39

in cacao, and showed that they present distinct expression profiles throughout fungal development and 40

infection. We determined the X-ray crystal structures of MpCP1, 2, 3 and 5, representative of different 41

branches of a phylogenetic tree and expressed at different stages of the disease. Structure-based 42

biochemistry, in combination with nuclear magnetic resonance and mass spectrometry allowed us to define 43

specialized capabilities regarding self-assembling and the direct binding to chitin and N-acetylglucosamine 44

(NAG) tetramers, a fungal cell wall building block, and to map a previously unknown binding region in 45

MpCP5. Moreover, fibers of MpCP2 were shown to act as expansin and facilitate basidiospore germination 46

while soluble MpCP5 blocked NAG6-induced defense response. The correlation between these roles, the 47

fungus life cycle and its tug-of-war interaction with cacao plants is discussed. 48

49

INTRODUCTION 50

The basidiomycete Moniliophthora perniciosa is the causal agent of the Witches’ Broom Disease 51

(WBD) and considered one of the major fungal plagues of cacao crops (Aime and Phillips-Mora 2005). 52

The WBD impacts severely the production of cocoa beans, the raw material of chocolate and other 53

derivatives, resulting in great agro-economic losses throughout the cultivating fields of Central and South 54

America (Evans 2007). In this regard it is urgent to gain knowledge on important proteins for the fungus 55

attack onto the plant. 56

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Defense responses in plants upon a pathogen attack usually result in deposition of callose, production 57

of reactive oxygen species, nitric oxide, phytoalexins, along with the transcription of defense-related genes 58

(Desender et al. 2007). These responses are promoted by elicitors, which are recognized by plant receptors. 59

They initiate the microbe-associated molecular pattern (MAMP)-triggered immunity or induce a second 60

layer of defense called the effector-triggered immunity (Jones and Dangl 2006). 61

One family of elicitors spread among the fungal kingdom is the cerato-platanin (CP) family. The 62

founding member of this family and the most studied protein is CP from Ceratocystis platani (Pazzagli et 63

al. 1999), hereon referred to as CpCP. CpCP, recently characterized as a MAMP, induces defense 64

responses and tissue necrosis in plants (Fontana et al. 2008, Lombardi et al. 2013, Pazzagli et al. 1999) and 65

may have a structural role in fungal cell wall formation (Baccelli et al. 2012, Boddi et al. 2004). The 66

tridimensional structure of CpCP was determined by NMR and is similar to proteins that bind to 67

polyssacharides (de Oliveira et al. 2011). The CpCP interacts with chitin (an unsoluble polymer of N-68

acetylglucosamine; NAG) and soluble NAG oligomers (Baccelli et al. 2013, de Oliveira et al. 2011), and 69

although does not bind to cellulose, CpCP loosens it, which could be important during host colonization 70

(Baccelli et al. 2013). Pop1, a CpCP homologue found in C. populicola, shares many of these features. 71

Indeed, it was demonstrated that both, CpCP and Pop1, form soluble ordered aggregates under mild 72

denaturing conditions, which are believed to be important for fungal development and virulence (Baccelli 73

et al. 2013, Carresi et al. 2006, Comparini et al. 2009, Lombardi et al. 2013, Martellini et al. 2012, Pazzagli 74

et al. 2009). Other cerato-platanins present diverse biological activities, such as the induction of systemic 75

acquired resistance (SAR) and induced systemic resistance (ISR) in plants, linked or not to tissue necrosis 76

(Djonović et al. 2007, Frías et al. 2011, 2013, Hall et al. 1999, Vargas et al. 2008, Wilson et al. 2002). Epl1 77

from the beneficial, root-colonizing fungus Trichoderma atroviride, was shown to be able to aggregate in a 78

quick and ordered fashion, although no biological role of such aggregates was presented (Frischmann et al. 79

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2013). The antigen CS-Ag from Coccidioides immitis, a human pathogen, has proteolytic activity (Pan and 80

Cole 1995). All these functions attributed to the CP proteins make them an attractive target to control the 81

pathogen attack. 82

The recent completion of M. perniciosa’s genome sequencing allowed us to identify an astonishing 83

high number of CP-coding genes, more precisely, 12 in total (hereon referred to as MpCP1 to 12). In this 84

manuscript, we show that they are clearly differentially expressed throughout the several stages of the 85

disease, as well as the different stages of the fungus development. Accordingly, the co-expressed proteins 86

clustered into distinct branches of an all-encompassing phylogenetic tree, indicating the presence of groups 87

with higher sequence similarity and likely functional proximity. In order to test this hypothesis, we 88

heterologoulsy expressed, purified and determine the crystallographic structure of four representative 89

members of the distinct phylogenetic clusters: MpCP1, which is exclusively expressed in basidiocarps, 90

MpCP2 and 3, both detected in the fast growing mycelium and necrotic infected seeds and fruits, and 91

MpCP5, majorly detected during the slow growth in the apoplast, where plant attack is set. Curiously, 92

MpCP5 structure was found to be essentially different from any other determined to date. The X-ray crystal 93

structures guided the subsequent investigation on MpCP’s capacity of binding to fungus and plant cell wall 94

components, as well as to self-aggregate into amyloid-like filaments. In this sense, NMR analysis showed 95

that MpCP3 and 5 distinctly bind to N-acetylglucosamine tetramers (NAG4) with different affinities and 96

allowed the description of an unforeseen binding interface in MpCP5. The MpCP5 presence was sufficient 97

to block NAG6-induced defense response in tobacco seedlings, representing the first CP shown to 98

counteract the chitin fragment plant sensitization. Finally, we show that MpCP2 and 3 are the most 99

responsive to self-aggregation into amyloid-like fibrils, with the fibrils of the former acting as expasins and 100

also promoting germination tube growth. MpCP5`s and MpCP2`s described biological activities are in 101

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accordance with their expression profile during the host-fungus interaction and exemplify the MpCP`s 102

protein function specialization during the Witches’ Broom disease of cacao. 103

RESULTS 104

M. perniciosa possesses multiple differentially expressed cerato-platanin genes - M. perniciosa has a 105

hemibiotrophic life cycle starting with meristem- and young fruits-infecting basidiospores that induce 106

hyperplasia and irregular branching of actively growing tissues of the cacao tree. During this stage, called 107

biotrophic phase, hyphae are formed in the host’s intercellular space. The disease then progresses to a 108

necrotic stage, during which the fungus grows intracellularly, resulting in the death of the plant tissues 109

(giving rise to the dry-broom aspect of the branches) and the development of fruiting bodies (necrotrophic 110

phase) (Meinhardt et al. 2008). 111

In addition to the five CPs originally described by Zaparoli and colleagues (2009) in the fungus M. 112

perniciosa (MpCP1 to MpCP5, NCBI Genbank accession codes EU250339, EU250340, EU250342, 113

EU250344 and EU250345, respectively), we present hereby seven new genes. It is yet worth mentioning 114

that the CERAT gene, previously identified by Rincones and colleagues (2008), corresponds to MpCP3. 115

An Aspf13 homologue was also described, and verified by us to match the MpCP2 gene (Leal et al. 2010). 116

Given the new apparent redundant number of isoforms (MpCP1 to 12), we first set out to analyze their 117

individual mRNA expression patterns throughout the different stages of the disease. RNA-seq data from 118

green-house grown cacao plants infected with M. perniciosa revealed that MpCPs 4, 5, 11 and 12 are 119

prominently expressed in the green parts-infecting fungus (biotrophic phase). Their gene expression levels 120

decrease as the disease progresses, until complete disappearance when the dry broom stage is reached (Fig. 121

1A). Secondly, MpCP2 and 3 are virtually the only two isoforms present in M. perniciosa from field-122

collected, infected fruits (Fig. 1B) and in the mycelium of in vitro lab-cultivated fungus (Fig. 1C). MpCP1 123

is basically the only isoform detected during basidiocarp formation, while MpCP4 and 11 dominated the 124

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basidiospore phase. MpCP6, 7, 8, 9 and 10 presented no significant gene expression throughout all the 125

samples and stages analyzed. Mass spectrometry analysis of M. perniciosa’s culture medium of in vitro 126

grown necrotrophic mycelium was further performed to allow the direct identification of the proteins. 127

Accordingly, MpCPs 2, 3 and 7 proteins (supplemental Table S1A) were identified, indicating that all other 128

MpCPs are likely only necessary during the fungus-cacao interaction. Glycosylation prediction using the 129

NetNGlyc 1.0 online server revealed that MpCPs 1, 2, 4, 5 and 11 contain one putative glycosylation site 130

(NxS/T), which is equivalent to the one described for the protein Sm1 as important to modulate protein 131

dimerization and plant recognition (29NGS31; Vargas et al. 2008). Additional putative glycosylation sites 132

were also identified for these MpCPs, which can be found in supplemental Table S1B. Two out of the three 133

spectrometry-identified MpCP2 peptides spaned the SM1-homologue glycosylation site indicating that, if 134

this protein is glycosylated on this segment, it is only partially. 135

Phylogenetic analysis of M. perniciosa’s cerato-platanin proteins - The clear differential expression 136

profiles of MpCP transcripts leaded us to speculate whether these isoforms might have distinct roles during 137

the fungus development or the plant infection process. In order to evaluate the evolutionary relationships 138

between the homologous sequences and gain insights on their specific roles, we reconstructed the cerato-139

platanin phylogenetic tree using deposited homologous sequences (Fig. 2). The group of sequences 140

included recently deposited genes from other cacao-infecting fungus, Moniliphthora roreri (MrCP1 to 10), 141

as well as the M. perniciosa biotype S, which infects plants of the Solanaceae family (Marelli et al. 2009) 142

(SMpCP1 to 3 and 6 to 12), and the saprotrophic Crinipellis campanella (CcCP1 to 6), representing a 143

saprotrophic non-pathogenic ancestral of the Moniliophthora genus (Aime and Phillips-Mora, 2005). The 144

sequences clustered in three major groups. One group contains CP homologues from ascomycetes and is in 145

accordance with previously published data (Seidl et al. 2006), regarding the clustering of the genes Sm1 (T. 146

virens), Epl1 (Hypocrea atroviridis), SP1 (Leptosphaeria maculans) and Snodprot1 (Phaeosphaeria 147

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nodorum), among others, within the so-called Epl1/Epl2 cluster (branches within light red box in Fig. 2). 148

MpCP4, 5 and 11, as well as homologues from the basiodiomycetes Pleurotus ostreatus and Coprinopsis 149

cinerea determined the MpCP Cluster 1 (Mper1; blue box in Fig. 2). On the other hand, MpCP2, 3, 7, 8, 10 150

and 12 grouped within the MpCP Cluster 2 (Mper2; green box in Fig. 2) along with saprotrophic C. 151

campanella’s CPs. 152

Mper clusters 1 and 2 are relatively distant in the evolutionary tree and, in accordance, comprise 153

MpCP proteins that are co-expressed during different stages of the fungus development. Specifically, 154

MpCP4, 5 and 11 (Mper1) are co-expressed during the fungus-cacao interaction (in the green/living parts 155

of the plant) as well as during basidiospore germination, while MpCP2 and 3 (Mper2) are highly expressed 156

only in the necrotic infected fruit and in vitro grown mycelium. Lastly, MpCP9, not significantly expressed 157

in any of the cases analyzed, clustered with several putative CP homologues from basidomycetes. MpCP1 158

and 6 did not significantly cluster into the cited groups. 159

This separation may suggest that MpCP4, 5 and 11 are important for the fungus-plant interaction 160

process and that MpCP2 and 3, as well as MpCP1, expressed in the basidiocarp, may have important 161

developmental roles for the growth in a nutrient-rich environment. The combination of the above 162

mentioned data led us to conclude that the CPs found in the M. perniciosa genome may have different roles 163

important for distinct aspects of the WBD. 164

X-ray structures - To unravel the different functions of the MpCPs, we solved the crystal structure of 165

selected recombinant MpCPs expressed during the infection of green parts (MpCP5) and fruits (MpCP2 166

and 3), as well as MpCP1, the major isoform during basidiocarp formation. Different constructs of a 167

recombinant MpCP4 were also tested for solubility after E. coli expression, but all failed in producing 168

soluble protein. The parameters and statistics for both data collection and processing and the final refined 169

models are presented in supplemental Table S2. 170

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First, MpCP1 structure was solved by single-wavelength anomalous dispersion using the signal from 7 171

intrinsic sulfur atoms (5 cysteine and 2 methionine residues). The obtained model was then employed as 172

search model for solving MpCP2 and MpCP3 structures by molecular replacement. MpCP5 could only be 173

solved by molecular replacement using the elicitor Sm1 from T. virens (PDB ID: 3M3G) as search model. 174

Regardless, the core fold of all MpCPs consists of a six-stranded antiparallel β-sheet flanked by six α-175

helices and loops, forming a double Ψβ-barrel (Fig. 3A). Also, all four structures presented the two 176

disulfide bridges expected for this family of proteins (supplemental Fig. S1). The overall MpCP1, 2 and 3 177

structures are highly conserved, with an average backbone r.m.s.d of 0.86 Å, and an average sequence 178

identity of 49.4%, as calculated by Coot (Emsley et al. 2010) (Fig. 3A). The major conformational 179

variability between these three structures reside in a β-hairpin loop between the strands 3 and 4, along with 180

some small divergences among other loops. MpCP5 sequence is only 25% identical in average to MpCP1, 181

2 and 3, and this is reflected in an overall higher core r.m.s.d. of about 1.33 Å. MpCP5 has an extra α-helix 182

(labeled as α2*), and the β-strand β6 previously identified on the other MpCPs breaks down into two 183

shorter β-strands (termed β6 and β6*). Moreover, MpCP5 presents a shorter β-hairpin between the strands 184

3 and 4 along with some other major conformational variability among the loops (Fig. 3B). Five strong 185

peaks (over 7σ in height) were observed in the Fourier difference electron density map for MpCP1, which, 186

with respect to their contributions to the dispersive component of the scattering factor, were interpreted as 187

zinc, sodium, and chloride ions (supplemental Fig. S2). All of these ionic species are found in high 188

concentration in the crystallization solution, with the exception of the chloride which was present in the 189

protein purification solution. No other extraneous density was found in the electron density maps of 190

MpCP2, 3 and 5. 191

All the structures were analyzed using the DALI server (Holm and Rosenström 2010), as a mean of 192

identifying similar folds and infer about their functions. Using a Z-score cutoff of 10, the best structural 193

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matches for all MpCPs were the elicitor Sm1 from T. virens (PDB ID: 3M3G) and CpCP from 194

Ceratocystis platani (PDB ID: 2KQA). Overall, all MpCPs superposed well to both Sm1 and CpCP, with 195

r.m.s. deviations ranging between 1 and 1.3 Å for the Cα, indicating very high structural conservation 196

between them. Still, by the same cutoff criteria, the DALI search identified two expansins, the bacterial 197

EXPB1 (Yennawar et al. 2006) (PDB ID: 2HCZ) and EXLX1 from corn plant (Kerff et al. 2008) (PDB ID: 198

3D30), as well as the pollen allergen Phlp1 from the grass Phleum pratense (unpublished data) (PDB ID: 199

1N10). CpCP was shown to interact with polymeric chitin oligomers of NAG, a sugar constituent of fungus 200

cell wall (Baccelli et al. 2013, de Oliveira el at. 2011). EXPB1 binds to maize cell walls, most strongly to 201

xylans, causing loosening of the plant cell wall (Yennawar et al. 2006). EXLX1 binds to cellulose and 202

peptidoglycan, but it lacked catalytic activity against these substrates (Kerff et al. 2008). Therefore, the 203

structural similarities clearly suggest that the MpCPs may bind sugar molecules and that this interaction 204

may be important for the plant-pathogen interaction. 205

NMR titration shows that NAG4 binds to distinct sites of MpCP3 and 5 - We next tested the ability of 206

the MpCP1, 2, 3 and 5 to binding NAG monomer and tetramer (NAG4), as well as determined the 207

dissociation constants of these sugars, by NMR spectroscopy. NAG monomer at 10 mM and a range of 208

NAG4 concentrations, from 100 µM to 10 mM, were titrated against 50 µM of 15N-labelled MpCP1, 2, 3 209

and 5, while monitoring the chemical shift and line width changes in the 15N-HSQC spectra. Even a 200 210

times molar excess of NAG did not cause any perturbations to the 15N-HSQC spectra of all tested MpCPs, 211

(supplemental Fig. S3A). However, when tested in the presence of NAG4, both 15N-MpCP3 and 15N-212

MpCP5 spectra were clearly perturbed. No significant changes were observed in the spectra of 15N-213

MpCP1, while 15N-MpCP2 was slightly disturbed in the presence of NAG4 (supplemental Fig. S3B). 214

By triple-resonance experiments we identified the NAG4-binding surface and calculated the binding 215

affinity to NAG4 for both MpCP3 and MpCP5. Peaks corresponding to residues Phe32, Thr34, Ala36, 216

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Cys37, Asp39, Trp68 and Asn69 in MpCP3, are those which have undergone the most perturbation upon 217

NAG4 binding (Fig. 4A, left panel; supplemental Fig. S3C), based on a chemical shift cut-off criteria of ∆δ 218

(15N+1H) ≥ 0.05 ppm. These residues are part of a region that is structurally similar to the one previously 219

described for CpCP (de Oliveira et al. 2011) and lies mainly within the helix α2 and the loops between 220

helices α2 and α3 and β-strands β2 and β3. The estimated Kd for MpCP3:NAG4 interaction, based on the 221

chemical shifts of eight consecutives residues, according to previously published methodology (Lehotzky 222

et al. 2010), was 24.7 ± 12.8 mM (Fig. 4B, upper panel). Strikingly, the MpCP5 peak assignment showed a 223

rather extended, completely unforeseen, NAG4-interacting surface on this protein. The mapped region, as 224

judged by chemically shifted and signal-faded (cut-off of -0.5) peak residues, is confined mainly at the C-225

terminus portion of MpCP5 (Fig. 4A left panel; supplemental Fig. S3D-E). Accordingly, NAG4 binds to 226

MpCP5 with a higher estimated affinity as judged by a Kd of 6.6 ± 1.7 mM (Fig. 4B, lower panel). These 227

results show that MpCP3 binds NAG4 through a conserved surface regarding the CpCP. MpCP5, on the 228

other hand, binds to NAG4 through an unforeseen surface and with a higher affinity. 229

To get a picture of the putative binding mode of NAG4 to MpCP3 and 5, we performed computational 230

blind docking. The MpCP3:NAG4 lowest energy model obtained showed a binding surface that matched 231

the residues disturbed by NAG4 interaction, as verified by the NMR experiments (Fig. 4A and 4C, left 232

panels). On the other hand, the MpCP5:NAG4 second lowest energy model indicated that NAG4 may bind 233

to part of the otherwise large disturbed surface measured by NMR. The results indicated that residues 234

Thr34, Ala36, Asp39, Trp68 from MpCP3 (Fig. 4C, left panel) and His92, Asn116 and Thr119 from 235

MpCP5, among others, (Fig. 4C, right panel) are likely directly involved in the sugar-protein interaction 236

through polar and non-polar contacts. To further confirm this finding, we double-mutated MpCP3 residues 237

Ala36 to a Thr and Asp39 to a Gly, as well as did a single Trp68 to Tyr mutation and tested the wild type 238

and mutant proteins for NAG4 binding capacity as judged by shifting on the proteins thermal melting 239

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points (supplemental Fig. S4). Accordingly, all the MpCP3 mutations affected their interaction to NAG4. 240

Similarly, we mutated MpCP5 Tyr88 to Ser, His92 to Ala, Asn116 to Lys and Thr119 to either Ser or Ala. 241

Only Asn116Lys mutation apparently increased protein affinity for NAG4, indicating that the 242

MpCP5:NAG4 binding surface is rather large and likely involves the simultaneous interaction of more than 243

one molecule. 244

MpCP5 blocked the perception of NAG6 by the plant. It has been reported that chitin fragments - 245

generated by the action of plant chitinases against the fungus cell wall during the early stages of infection - 246

can act as MAMPs, eliciting immune responses in plants (Boller 1995, de Jonge et al. 2010). MpCP5 is 247

expressed while the fungus is interacting with the living parts of the plant, when a host response to the 248

pathogen attack is expected. In order to evaluate the functional significance of the NAG oligomer-MpCP5 249

interaction we exposed tobacco plants to the chitin fragment NAG6 as well as NAG6 pre-incubated with 250

MpCP5, and investigated, by quantitative PCR, the expression of defense-related genes (Fig. 4D). As 251

expected, plant responded to the NAG6 presence by increasing expression of tpa1 (phenylalanine ammonia 252

lyase), involved on phytoalexin synthesis pathway and SAR activation (Gayoso et al. 2010), npr1 (non-253

expresser of PR genes 1), a transcription factor pivotal for the activation of SAR (Dong 2004), pr-4a and 254

pr-5 (pathogenesis-related) genes, both described as displaying antifungal activity (Guevara-Morato et al. 255

2010, van Loon et al. 2006). The pre-incubation of MpCP5 with the NAG6 fragments reduced the response 256

levels back to control (just buffer), suggesting that MpCP5 protein can help blocking plant defense 257

response by scavenging released chitin fragments. 258

The MpCPs neither have glycolytic activity against nor bind to plant cell wall components - Similarly 259

to CpCP and the bacterial expansin EXLX1, the MpCPs presents structural conservation with the catalytic 260

domain found in family 45 of glycosyl hydrolases and in the MltA family of lytic transglycosylases 261

(supplemental Fig. S5) (de Oliveira et al. 2011, Kerff et al. 2008). Nevertheless, tests for hydrolytic activity 262

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of MpCPs 1, 2, 3 and 5 against some common plant cell wall components and other polysaccharides, as 263

well as cacao cell wall extract (CCW), all proved negative (Fig. 5A). Given that MpCP3 and 5 were shown 264

to bind to the soluble tetrameric form of NAG, we questioned whether these proteins would be able to bind 265

to insoluble chitin polymers via pull-down assay, therefore mimicking a possible interaction with fungal 266

cell wall. The pull-down assays were also performed against the CCW. As described for the CpCP protein 267

(Baccelli et al. 2013), MpCP1, 2, 3 and 5 did not bind to the plant cell-wall (Fig. 5B) but they were all (on 268

both soluble - Fig. 5C - and aggregated forms - data not shown) pulled-down by chitin polymers, with 269

MpCP3 apparently binding the strongest. 270

MpCP2 and 3 have higher propensity to form soluble aggregates that induces Thioflavin T 271

fluorescence - The MpCPs-highly related protein CpCP resembles another class of fungal-secreted proteins 272

known as hydrophobins, which have many distinct roles in fungal development and in pathogen–host 273

interactions (Pazzagli et al. 2009, Whiteford and Spanu 2002). In this regard, we studied the MpCP1, 2, 3 274

and 5 self-assembling properties. A range of 0.1 mM up to 3 mM of each MpCP was incubated for 30 days 275

at 37°C under low pH treatment (pH 3.0), as previously described for the CpCP protein (Pazzagli et al. 276

2009), and the Thioflavin T (ThT) fluorescence signal monitored. The existence of a threshold 277

concentration below which aggregation does not occur is an important feature associated with amyloid 278

fibrils (Lomakin et al. 1996). As seen in Fig. 6A, the MpCP2’s ThT fluorescence readings drastically 279

increased (over 15 times in comparison to control) from 0.4 to 0.8 mM of protein concentration. MpCP3 280

and MpCP5, although to a smaller extent, also induced significant ThT fluorescence at 0.8 mM protein. In 281

comparison to the other analyzed MpCPs, MpCP1 showed low levels of ThT-positive aggregates at only 282

above 1.5 mM of protein concentration. The initial aggregation step is commonly known to start from a 283

partly unfolded monomeric intermediate which presents increased surface hydrophobicity (Murphy 2007). 284

Accordingly, the fluorescence intensities of the probes 1,8-ANS and bis-ANS, two hydrophobic dyes that 285

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bind to the solvent-exposed hydrophobic surfaces were greatly enhanced upon interaction with 0.8 mM 286

MpCP1, 2, 3 and 5 aggregates (Fig. 6B). 287

In light of these results we performed a time course study of protein aggregation of 0.8 mM of MpCP2 288

and MpCP3, at 37°C in low pH solution. The kinetic curves of the ThT fluorescence intensity are 289

consistent with a nucleation-dependent polymerization model commonly observed for proteins that can 290

form amyloid fibrils (Lomakin et al 1996). While MpCP2 took 3 days to reach a signal plateau (Fig. 6C), 291

MpCP3 showed similar pattern, but only after 25 days of incubation. 292

Biophysical and functional characterization of the soluble aggregates - The secondary structure 293

changes accompanying MpCPs aggregation (at a protein concentration of 0.8 mM) were analyzed by far-294

UV circular dichroism (CD) spectroscopy after 5, 10 and 20 days of low pH incubation at 37 °C. In as early 295

as 5 days, it was possible to detect a loss of the MpCP1 native conformation and the acquisition of a 296

random coil structure, with a negative band at 200 nm (Fig. 7A), suggesting protein unfolding, as further 297

confirmed by NMR spectroscopy (supplemental Fig. S6, left panel). Curiously, the highly ThT-responsive 298

MpCP2, as well as MpCP3 and 5, did not present characteristic random-coil spectra, neither a shift from α-299

helix to β-sheet content, as commonly found in β-amyloid fibers. This is somewhat in agreement with a 300

previous report, in which the CpCP did not display conformational changes corresponding to an increase in 301

β-structure, being rather unfolded or acquiring a new secondary structure pattern (Pazzagli et al. 2009). 302

Globular proteins under equilibrium conditions may have at least two different partially folded 303

conformations, the molten globule and its precursor, the pre-molten globule, both of which may potentially 304

play a role as the crucial amyloidogenic species (Uversky and Fink 2004). The most characteristic feature 305

of the amyloidogenic pre-molten globule CD spectra is a large negative ellipticity at 200 nm and low 306

ellipticity in the vicinity of 222 nm, with native/molten globule presenting combination of opposite 307

situations and the unfolded proteins showing the largest negative values at 200 nm and ellipticity in the 308

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vicinity of 222 nm close to zero (Uversky and Fink 2004). According to a survey of CD spectra of several 309

proteins captured on different states, the MpCP2, 3 and 5 CD profiling, as wells as their MRE[θ]200 x 310

MRE[θ]222 combination, shows that the mild denaturing treatment likely brought the protein to a molten 311

globule state (Fig. 7B), while MpCP1 positions closer to coordinates more characteristic of an unfolded 312

state. 313

Dynamic light scattering analysis of MpCP1, 2, 3 and 5 before and after 15 days of incubation in low 314

pH solution confirmed the formation of aggregates of protein (Fig. 7C). Since MpCP5 was one of the 315

proteins with the smallest ThT signal we further characterized its aggregation process by RMN. Two-316

dimensional 15N-HSQC spectra indicated that, after 4 days of incubation at 37oC at low pH, the great 317

majority of peaks either had a decreased intensity or disappeared, a clear indication of oligomerization 318

process (Cavanagh et al. 2007) (supplemental Fig. S6, right panel). The filamentous nature of the 319

aggregates was visually confirmed for MpCP2, by using transmission electron microscopy (Fig. 7D). 320

The Ceratocystis platani CP self-aggregation correlates to a fragmentation process, so as for some 321

amyloid-like proteins which undergo self-assemblage after a cleavage of the polypeptide chain (Mishra et 322

al. 2007, Pazzagli et al. 2009, Tsiroulnikov et al. 2006). The identification of the cleavage sites and 323

aggregating peptides of MpCP1, 2, 3 and 5 was done by mass spectrometry and are in clear accordance 324

with what was described for CpCP (Pazzagli et al. 2009) (supplemental Fig. S7, S8 and Table S3). 325

Finally, we further investigated the biological relevance of the aggregation processes by assessing the 326

capability of aggregated MpCP2 of loosening cellulose. Soluble CpCP and Pop1 were shown to fragment 327

cellulosic substrates through a non-enzymatic mechanism (Baccelli et. al, 2013). We verified that 328

aggregated MpCP2, but not its soluble monomeric form, promotes cellulose fragmentation which may 329

implicate a role of this protein’s aggregates during the interaction of M.perniciosa with the cocoa (fixation 330

to the substrate and nutrient acquisition) (Fig. 7E). Aggregated MpCP2 also potentiated the growth M. 331

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15

perniciosa basidiospore’s germination tube (Fig. 7F), a phenomenon which may be related to the fungus’ 332

hyphal development. Curiously, as shown in Fig 1B and C, MpCP2 is highly expressed in the in vitro-333

grown mycelium and in the fungus of infected fruits, situations where a fast grown is propitiated by the 334

nutrient rich environment. 335

336

DISCUSSION 337

M. perniciosa, a fungus with a hemibiotrophic lifestyle, presents twelve copies of CP-coding 338

sequences in its genome. Different genetic evolutionary models predict an expansion in a gene’s copy 339

number before their specialization or the rise of new functions, also accounting for an overlap of functions 340

due to a momentary intermediate state of specialization (Bergthorsson et al. 2007). In this work we show 341

that the MpCPs (1 to 12) are expressed at particular stages of the Witches’ Broom disease, and, based on 342

our data and on the literature above, we reason that M. perniciosa evolved different proteins to perform 343

different, specialized roles during its hemibiotrophic life cycle. 344

The biotrophic stage of M. perniciosa is unusually extended, lasting for 2–3 months in the plant’s 345

infected tissue and creating a nutrient sink to the infection site, so that the host is disadvantaged but not 346

killed (Scarpari et al. 2005). During this stage, preventing the elicitation of the host’s immune response is 347

key for the fungus survival. None of the purified MpCPs caused necrotic symptoms or phytoalexin 348

induction on tobacco leaves injected with these proteins (supplemental Fig. S9), in accordance with results 349

obtained with CtCP1, the CP from Colletotrichum truncatum, another hemibiotrophic pathogen, which 350

protein also did not induce defense responses in planta (Bhadauria et al. 2011). This may suggest an 351

evolutionary pressure to reduce the defense-eliciting ability of CP proteins in hemibiothrophic life styles. 352

However, our findings are in contrast with a previous report (Zaparoli et al. 2009) which showed MpCP1`s 353

necrotic activity on tobacco and cocoa leaves. It is possible that the observed differences are due to the 354

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presence of bacterial contaminants on their sample, since we added a further purification step in our protein 355

preparation. 356

Gene transcripts of MpCP4, 5 and 11, from the cluster Mper1 of the presented phylogenetic tree, were 357

exclusively detected in the slow-growing biothrophic phase, when the fungus develops in the apoplast of 358

the plant and where the pathogen-host survival battle is established. Using NMR, we have shown that 359

recombinant MpCP5 binds to NAG4 with the highest affinity via a previously unidentified binding site. 360

This interface, in MpCP5, is located around a much shorter β-hairpin formed between sheets β3 and β4, 361

when compared to MpCPs 1 and 2. The large protein surface disturbed by NAG4 opens the possibility that 362

either the protein goes through a profound conformational change due to the sugar interaction or multiples 363

binding sites are existent. Gene expression data showed that MpCP5 is capable of blocking the plant 364

response defense to the chitin fragment NAG6, a potent defense elicitor released during fungal colonization 365

(Boller 1995). Taking together, these results create a potential link between MpCP5`s biotrophic phase 366

expression, its NAG oligomer-binding property and its capacity of suppressing the plant immune response. 367

Likewise, MpCP4 and 11 may also be important in protecting the fungus against the cacao, since most of 368

the key residues we identified for NAG4 binding in MpCP5 are conserved on these proteins. Thus far, the 369

CPs have been implicated as fungal elicitors and/or effectors, either promoting the virulence of pathogenic 370

fungi, or enhancing the plant’s resistance against other microorganisms (Djonović et al. 2007, Frías et al. 371

2011, 2013, Jeong et al. 2007, Pazzagli et al. 1999 Vargas et al. 2008). Although de Oliveira and 372

colleagues (2011) have hypothesized that CPs might act as defense suppressors by scavenging free chitin 373

fragments, we present hereby a experimental evidence which suggest such thing, bringing a potential new 374

feature to this class of protein. 375

The MpCPs belonging to the phylogenetic cluster Mper2 (MpCP2, 3, 7, 8, 10 and 12) positioned 376

together with homologues from the saprothrophic C. campanella and their gene transcripts were detected 377

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during the late and necrotic stages of the cacao fruits infection, as well as in the in vitro grown mycelium. 378

Worth of note, MpCP3 was previously detected in our laboratory in an in vitro-grown biotrophic-like 379

mycelium (Rincones et al. 2008), and the gene fragment was identified as CERAT by then. Even though 380

the biotrophic-like mycelium used by Rincones and colleagues presented biotrophic morphological 381

characteristics (thick, monokaryotic hyphae without clamp connections), further studies in our laboratory 382

showed that it also expressed some of the genes typically found in the necrothrophic stage of the disease 383

(data not shown), probably due to the use of cocoa extract-containing conditions (nutrients which are 384

usually only available during necrotrophic phase). 385

NMR data showed that MpCP3 also binds to NAG4, however with a lower affinity when compared to 386

MpCP5. MpCP3 shares a similar NAG4-binding surface to the one previously described for CpCP as 387

confirmed by site-directed mutagenesis and thermal shift assays. Computational docking allowed the 388

determination of an interface involving the diametrically opposed loops between β2 and β3 (which is much 389

larger than its equivalent in MpCP5), and helices α1 and α2. The sequence composition of these loops in 390

MpCP3 is not only distinct from those in MpCP5, but it also diverges between MpCP1 and 2, thus 391

explaining why the later two did not bind significantly to NAG4. The three residues directly involved in 392

MpCP3 binding to NAG4 (Ala36, Asp39 and Trp68) are fully conserved on both MpCP7 and 9, suggesting 393

that these proteins also bind to NAG4. Worth of noting, similarly to MpCP3, MpCP7 and 9 are expressed 394

on the rotten infected fruits/plant and lab-cultivated fungus, both representative of the fast-growing 395

necrotrophic stage. 396

The CPs from C. platani and C. populicola have been shown to self-aggregate under mild-denaturing 397

conditions, such as acetic acid and TFE solution, as well as exposure to hydrophobic surfaces (Martellini et 398

al. 2012, Pazzagli et al. 2009). The protein Epl1 presented spontaneous quick assembling but no further 399

characterization of the aggregates was performed (Frischmann et al. 2013). Another protein family present 400

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in fungi that is able to enhance ThT fluorescence after its assemblage is the hydrophobins (HPB) (Wösten 401

and de Vocht, 2000). Rodlets formed by aggregated hydrophobins first lower the surface tension at the 402

water–air interface and are important for fungal development and virulence by creating amphipathic layers 403

that facilitate hyphal growth and adhesion, as well as the formation of haustoria and fruiting bodies. 404

Similarly to the CpCP and MpCPs, HPBs have intramolecular disulfide bonds, are secreted as monomers 405

and self-aggregate under favorable conditions (Whiteford and Spanu 2002). All four tested MpCPs were 406

capable of self-aggregation after incubation in low pH solution. MpCP1, the one with the slowest 407

aggregation kinetics, presents levels comparable to CpCP (Pazzagli at al. 2009), while MpCP2, followed 408

by MpCP3, are the most responsive, as qualitatively judged by the kinetics curves of ThT fluorescence. 409

In physiological environments, where many processes are dictated by interface phenomena, the 410

presence of a surface (such as the host cuticle, cell wall and/or membrane) may provide the necessary 411

perturbation to induce the conversion from the soluble conformation to the self-assembling conformation 412

(Martellini et al. 2012, Moores et al. 2011, Murphy 2007). The native globular structure of several proteins 413

has been shown to be destabilized to pre-molten/molten globule-like structures by a myriad of different 414

agents, e.g. high temperatures, organic solvents, low or high pH, low to mild concentrations of strong 415

denaturants, and others, which may significantly accelerate the rate of fibril formation (Uversky and Fink, 416

2004). Another aspect of CpCP and other aggregating proteins mirrored by the MpCPs is the self-cleavage 417

process as a prerequisite for the process (Mishra et al. 2007, Pazzagli et al. 2009, Tsiroulnikov et al. 2006), 418

probably enabling the exposure of hydrophobic regions of the protein that can then establish the first 419

intermolecular contacts needed for self-assembling. 420

We propose that the chitin binding property of both MpCP2 and MpCP3, along with their and MpCP1 421

self-aggregation characteristic, all proteins expressed during fast-growing phases of the fungus, very likely 422

facilitate the process of hyphal growth, fruiting body formation and substrate adhesion. Furthermore, 423

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MpCP2 aggregates, but not the soluble protein, revealed expansins characteristics as it was able to promote 424

cellulose fragmentation. Plant cell wall (rich in cellulose) loosening activity may be important for host 425

colonization during fungal infection, but also for growth on dead plant tissue and nutrient acquisition. 426

Moreover, MpCP2 aggregates were also shown to contribute to germination tube formation and are likely 427

to be important for the fungus development. Although not tested, MpCP3 binding surface to NAG4 (which 428

is distinct to MpCP5 surface) may be important for the chitin binding (this isoform was the one with higher 429

qualitative affinity to chitin), fungus cell wall remodeling and growth. 430

M. perniciosa presents a well-characterized, yet complex, lifestyle (Meinhardt et al. 2008). The 431

existence of a biotrophic phase, followed by the progression to a necrotrophic stage, requires the fungus to 432

thrive at the most diverse physiological environments inside its host. Consistently, we propose, for the first 433

time, a differentiation model for cerato-platanins in an organism, based on protein functional specialization. 434

M. perniciosa is the only basidiomycete phytopathogen among all previously studied CP-bearing 435

organisms and, overall, the results provided in this work may help constitute a useful platform for 436

compound design which can block the protein activity and help to compose a combat approach against the 437

disease. 438

439

MATERIALS AND METHODS 440

Genome survey - To identify Cerato-platanin orthologous genes in the M. perniciosa biotype C 441

genome (Mondego et al. 2008) we searched for sequences classified within the InterPro ID IPR010829 442

(Hunter et al 2012). Five genes (MpCP1 to 5) had been previously described (Zaparoli et al. 2009). Seven 443

new ones were further identified and named MpCP6 to MpCP12. The open reading frames (ORFs) of all 444

MpCP from M. perniciosa biotype C genes were confirmed by cDNA sequencing and deposited at the 445

GenBank (refer to the first page and the results for accession numbers). To identify the CP’s sequences 446

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from the isolate MCA2977 of M. roreri and the isolate APS1 of M. perniciosa biotype S, we used genomic 447

and RNA-seq data (unpublished data). CP’s introns were found through the alignment between RNA-seq 448

and genomic derived sequences of the M. perniciosa biotype S and M. roreri. Since there is no expression 449

data for the C. campanella, its CP`s genomic sequences were evaluated for intron presence with the aid of 450

the software Augustus Gene Finder v2.3 (Stanke et al. 2004) trained with genes of C. cinerea and ESTs 451

from M. perniciosa, as previously described (Mondego et al. 2008). The software SignalP 4.0 (Petersen et 452

al. 2011) was used to predict the presence of secretion signal peptide in all of the encoded proteins. 453

RNA-seq - The reproductive structures of M. perniciosa (basidiomata) were produced following the 454

procedures described (Pires et al. 2009) and were used to collect basidiospores directly in the collection 455

solution (16% glycerol, 0.01M MES, 0.01% Tween, pH 6.1) as described elsewhere (Frias et al. 1995). 456

Theobroma cacao var. Comum was cultivated in a greenhouse under controlled temperature and humidity 457

conditions (from 22 to 28 °C and above 50%, respectively). Two-month old seedlings were infected with 458

Moniliophthora perniciosa basidiospores as previously described (Frias et al. 1995) and RNA was 459

extracted from infected parts of the plant at representative time-points of the disease, from the green broom 460

to complete necrosis. Infected fruits from Theobroma cacao trees var. Comum were collected in a cacao 461

farm located in Ilhéus, Brazil. Three stages of the disease were analyzed: early stage (which is 462

characterized by disordered fruit ripening), partial necrosis stage and rotten fruit stage. The seeds and pods 463

of each fruit were collected independently for RNA extraction. For RNA extraction of M. perniciosa 464

basidiospores, 1 mL of a suspension containing approximately 107 spores/mL was gently centrifuged at 465

1000xg for 5 minutes to precipitate the basidiospores, which were resuspended in 1 mL collection solution 466

(which prevents the germination), or ressuspended in 1 mL of sterile water (to allow germination). Both 467

aliquots were transferred to small beakers, and incubated at 28°C and 120 rpm for 4 hours. The 468

germination of the basidiospores was microscopically verified prior to RNA extraction. In vitro grown 469

mycelium or mature basidiomata were pooled and used for RNA extraction. 470

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For each sample, 10 µg total RNA were used to prepare the mRNA-seq library according to the 471

protocol provided by Illumina. Library quality control and quantification were performed with the Experion 472

Automated Electrophoresis System (Bio-Rad) and the Qubit fluorometer (Invitrogen), respectively. Each 473

library was sequenced in one lane of an Illumina Genome Analyzer IIx sequencer to produce 36 bp single-474

end reads. The RNA-seq reads were aligned against a reference comprised of 17.008 gene models of M. 475

perniciosa using the program Bowtie (Langmead et al. 2009). The alignment was performed by allowing 476

up to two mismatches, and excluding reads that mapped to more than one position in the reference. The 477

number of reads mapped to each gene was divided by the size of the gene (in kilobases) and then 478

normalized by the total mapped reads of the library (in millions of reads). Thus, the expression value of 479

each gene was given in RPKM (mapped reads to a gene per kilobase per million of total mapped reads), so 480

that the level of expression of genes within the same library and in different libraries are comparable 481

(Mortazavi et al. 2008). This data is part of the WBD Transcriptome Atlas (unpublished data). 482

Heterologous MpCPs production - MpCP coding genes were amplified from a cDNA library 483

prepared from M. perniciosa as described elsewhere (Zaparoli et al. 2009). The constructs were cloned into 484

a modified version of pETSUMO (Invitrogen) and transformed into E. coli Origami 2 (Merck) chemically 485

competent cells. The final constructs spanned residues 20–145 (MpCP1), 17–140 (MpCP2), 15–135 486

(MpCP3), and 22–156 (MpCP5). For MpCPs purification, the transformed bacteria were lysed and the 487

clarified soluble fraction purified by a two-step procedure, starting with Immobilized Metal Ion Affinity 488

Chromatography (IMAC) using the Co2+-charged TALON resin (BD Biosciences), equilibrated with 50 489

mM Tris-HCl pH 8.5, 150 mM NaCl. The resin was washed extensively with this solution and then 490

incubated overnight at room temperature with appropriated amount of the protease ULP-1 for His-SUMO 491

tag removal. The cleaved protein was eluted from the resin and loaded onto the Superdex 75 HR 10/30 gel 492

filtration column (GE Healthcare) equilibrated in 50 mM Tris-HCl, pH 8.5 and 150 mM NaCl. Several 493

attempts to produce soluble MpCP4 did not succeed well. The eluted protein was concentrated to the 494

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required concentration as judged by UV280nm absorption and calculated coefficient extinction value as 495

estimated through the primary sequence and the ExPASy ProtParam tool (Gasteiger et al. 2005), and used 496

for crystallization screens as well as functional assays. 497

X-ray crystallography - Crystals were grown using the conventional sitting drop, vapor diffusion 498

method in the following conditions: MpCP1 - 0.1 M sodium acetate pH 4.5, 20% PEG 1000, 0.2 M zinc 499

acetate; MpCP2 - 0.1 M sodium acetate pH 4.6, 8% PEG 4000; MpCP3 - 0.1 M bis-tris propane pH 7.0, 500

1.5 M ammonium tartrate; MpCP5 - 0.1 M sodium acetate pH 5.5, 5% PEG 400, 18% PEG 3350. X-ray 501

diffraction datasets were collected at beamlines D03B-MX1 and W01B-MX2, at the Brazilian National 502

Synchrotron Laboratory, LNLS. All data were integrated using Mosflm (Leslie 1992) and scaled with 503

SCALA (Evans 2006). MpCP1 was solved by Sulfur-SAD using SHELX (Sheldrick 2010) and the first set 504

of phases of MpCP2 and MpCP3 was obtained by the molecular replacement with MrBUMP (Keegan and 505

Winn 2008), using the MpCP1 monomer as the search model. The initial set of phases of MpCP5 was 506

obtained using Phaser (McCoy et al. 2007) and the protein Sm1 from T. virens as the search model (PDB 507

ID: 3M3G). Positional and B-factor refinement cycles, as well as solvent modeling, were performed with 508

Refmac (Vagin et al. 2004) followed by visual inspection using COOT (Emsley et al 2010). 509

NMR spectroscopy - 15N- and 13C/15N-labelled MpCP proteins were produced and purified as 510

described in supplemental material, dialyzed against a solution containing 20 mM Na2HPO4-NaH2PO4 pH 511

7.2, 50 mM NaCl and then added of 10% D2O for NMR experiments. NMR spectra were recorded on a 512

Varian Inova 600-MHz spectrometer equipped with a cryogenic probe. The spectra were processed with 513

NMRPipe/NMRDraw (Delaglio et al. 1995) and analyzed with NMRView (Johnson and Blevins 1994). 514

The backbone assignment of MpCP3 and 5 were determined using the three-dimensional experiments 515

HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO (Ikura et al. 1990, Grzesiek and Bax 1993, Wittekind and 516

Mueller 1993, Yamazaki et al 1994) and the graphical interface Smartnotebook (Slupsky et al. 2003). 517

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Titrations of 15N-labeled MpCPs proteins with NAG and NAG4 were followed by the recording of the two-518

dimensional 15N-HSQC spectra. For more details on the techniques describes and other techniques, see 519

supplemental material. 520

The following procedures are described in the Supplemental Material: Phylogenetic reconstruction; 521

Crystallization, X-ray Crystallography Data Collection and Processing; Structure Solution and Refinement; 522

NMR sample preparation; N-acetylglucosamine titration NMR experiment; N-acetylglucosamine docking 523

to MpCP3 and MpCP5, mutagenesis and fluorescence-based thermal shift assays; qPCR; Cacao cell wall 524

extraction; Pull-down assays; Thioflavin T, 1,8-ANS and bis-ANS fluorescence measurements; Cellulose 525

fragmentation; Spore germination; Circular dichroism spectroscopy; Identification of native MpCPs by 526

mass spectrometry; Transmission Electron Microscopy; Studies of protein cleavage associated to self-527

assembling by mass spectrometry; Injection of MpCPs in tobacco leaves. 528

ACKNOWLEDGEMENTS 529

This work was supported by FAPESP grant 2010/51884-8 (to ALBA), FAPESP fellowships 530

2010/14504-2 (to MROB) and 2010/51891-4 (to JFO) and CNPq fellowship 400796/2012-0 (to AC). We 531

thank LNBio for financial support and access to all facilities (MAS, LPP, LEC, Robolab and NMR). We 532

also thank LNNano for accessibility to LME, as well as the staff of the X-ray crystallography beamlines 533

(D03B-MX1 and W01B-MX2, at LNLS) used in this work. We thank Dr. Alessandra Girasole for technical 534

support and Gabriel Lorencini Fiorin for promptly providing fungal material used in this study. 535

536

AUTHOR CONTRIBUTIONS 537

MROB performed heterologous protein production and purification, polysaccharide interaction 538

assays, basidiospore germination assays, biophysical and biochemical characterizations, in planta defense 539

response experiments and edited the manuscript. JFO performed heterologous protein production and 540

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purification, X-ray crystallography and structure refinement, biophysical and biochemical 541

characterizations, NMR experiments, identified NMR spectra peaks and edited the manuscript. DAM 542

performed phylogenetic relationship construction and analysis, and executed qPCR experiments. PJPLT 543

and GAGP designed and performed RNAseq experiments. PFVP and HOT performed heterologous protein 544

production and purification. MLS and ACMZ designed, performed and interpreted NMR experiments. AC 545

and RVP performed transmission electron microscopy of MpCP2. PSLO performed docking experiments. 546

SMGD designed biophysical and biochemical characterizations, interpreted the data and edited the 547

manuscript. ALBA designed biophysical and biochemical characterizations, designed and performed X-ray 548

crystallography and structure refinement, interpreted the data and edited the manuscript. 549

550

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(EXLX1), a bacterial expansin that promotes root colonization. P. Natl. Acad. Sci. USA 105, 16876–647

16881. 648

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J., Cotomacci, C., Carraro, D.M., Cunha, A.F., Carrer, H., et al. (2008). A genome survey of 681

Moniliophthora perniciosa gives new insights into Witches’ Broom Disease of cacao. BMC Genomics 682

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solution properties of rigid proteins from atomic and residue-level models. Biophys. J. 101, 892-898. 691

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Ceratocystis fimbriata f. sp. platani. J. Biol. Chem. 274, 24959–24964. 696

Pazzagli, L., Zoppi, C., Carresi, L., Tiribilli, B., Sbrana, F., Schiff, S., Pertinhez, T.A., Scala, A. 697

& Cappugi, G. (2009). Characterization of ordered aggregates of cerato-platanin and their involvement 698

in fungus-host interactions. Biochem Biophys Acta 1790, 1334–1344. 699

Petersen, T.N., Brunak, S., Von Heijne, G., & Nielsen, H. (2011). SignalP 4.0: discriminating 700

signal peptides from transmembrane regions. Nat. Methods 8, 785–786. 701

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Porto, R.F., Villela-Dias, C., Brendel, M., Cascardo, J.C.M., et al. (2009). Early development of 703

Moniliophthora perniciosa basidiomata and developmentally regulated genes. BMC Microbiol. 9, 158. 704

Rincones, J., Scarpari, L.M., Carazzolle, M.F., Mondego, J.M.C., Formighieri, E.F., Barau, J.G., 705

Costa, G.G.L., Carraro, D.M., Brentani, H.P., Vilas-boas, L.A., et al. (2008). Differential Gene 706

Expression Between the Biotrophic-Like and Saprotrophic Mycelia of the Witches’ Broom Pathogen 707

Moniliophthora perniciosa. Mol. Plant Microbe In. 21, 891–908. 708

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disease of cocoa in Brazil caused by Crinipellis perniciosa. J. Exp. Bot. 56, 865–877. 711

Seidl, V., Marchetti, M., Schandl, R., Allmaier, G. & Kubicek, C. (2006). Epl1, the major 712

secreted protein of Hypocrea atroviridis on glucose, is a member of a strongly conserved protein family 713

comprising plant defense response elicitors. FEBS J. 273, 4346–4359. 714

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importance of being unfolded. Biochem. Biophys. Acta 1698, 131–153. 725

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Vargas, W., Djonović, S., Sukno, S. & Kenerley, C. (2008). Dimerization controls the activity 731

of fungal elicitors that trigger systemic resistance in plants. J. Biol. Chem. 283, 19804–19815. 732

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Mo.l Plant Pathol. 3, 391–400. 734

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secreted protein from Leptosphaeria maculans, the blackleg pathogen of Brassica napus. Mol. Plant 736

Pathol. 3, 487–493. 737

Wittekind, M. & Mueller, L. (1993). HNCACB, a High-Sensitivity 3D NMR Experiment to 738

Correlate Amide-Proton and Nitrogen Resonances with the Alpha- and Beta-Carbon Resonances in 739

Proteins. J. Magn. Reson. Ser. B 101, 201–205. 740

Wösten, H. & de Vocht, M. (2000). Hydrophobins, the fungal coat unravelled. Biochim. 741

Biophys. Acta 1469, 79–86. 742

Yamazaki, T., Lee, W., Arrowsmith, C., Muhandiram, D.R. & Kay, L.E. (1994). A Suite of 743

Triple Resonance NMR Experiments for the Backbone Assignment of 15N, 13C, 2H Labeled Proteins 744

with High Sensitivity. J. Am. Chem. Soc. 116, 11655–11666. 745

Yennawar, N., Li, L., Dudzinski, D., Tabuchi, A. & Cosgrove, D. (2006). Crystal structure and 746

activities of EXPB1 (Zea m 1), a beta-expansin and group-1 pollen allergen from maize. P. Natl. Acad. 747

Sci. USA 103, 14664–14671. 748

Zaparoli, G., Cabrera, O., Medrano, F., Tiburcio, R., Lacerda, G. & Pereira, G. (2009). 749

Identification of a second family of genes in Moniliophthora perniciosa, the causal agent of Witches’ 750

broom disease in cacao, encoding necrosis-inducing proteins similar to cerato-platanins. Mycol. Res. 751

113, 61–72. 752

753

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FIGURES CAPTIONS 754

FIGURE 1. Differential expression of MpCP1 to 12 throughout the stages of fungus development and 755

infection. (A) Out of 12 CP-coding genes identified in M. perniciosa, only transcripts from isoforms 4, 5, 756

11 and 12 are detected in the biotrophic fungus. A decrease in their expression levels is observed as the 757

disease progresses, until complete depletion when the dry-broom stage is reached. (B) Inversely 758

proportional levels MpCP2 and 3 coding transcripts are measured when rotten, field-collected seeds and 759

fruit pods are analyzed. (C) In samples extracted from lab-cultivated fungus, MpCP2 and 3 transcripts are 760

detected in the mycelium, whereas isoforms 4 and 11 are exclusively detected in germinated basidiospores. 761

MpCP1 is only detected in basidiocarps. Asterisks indicate isoforms that were detected at protein level, by 762

mass spectrometry, in the culture medium of in vitro grown necrotrophic mycelium. RPKM, Reads Per 763

Kilobase of exon model per Million mapped reads. 764

FIGURE 2. Phylogenetic reconstruction of the Cerato-platanin family. Comprehensive evolutionary tree 765

determined using deposited CP sequences. Four main clusters can be distinguished. Epl1/Epl2 (red box), in 766

accordance to previously published analysis (Seidl et al 2006); Mper1 and Mper2, delimited by blue, and 767

green boxes, respectively; and the sequences from other basidiomycetes, together with MpCP9 (not 768

highlighted). Numbers near branches are Bayesian posterior probabilities. 769

FIGURE 3. Crystal structures of MpCP1, 2, 3 and 5. Stereographic views in cartoon representations of 770

superposed MpCP1-3 (A) and MpCP1 and MpCP5 (B). All four structures present the canonical double 771

Ψβ-barrel, interspersed with helices. The major structural differences between MpCP5 and MpCP1, 2 and 3 772

is the length of the loop between sheets β2 and β3 and the hairpin connecting sheets β3 and β4. 773

FIGURE 4. MpCP3 and 5 bind to NAG4, though at distinct regions. (A) Overlay of regions of 15N-HSQC 774

spectra of 15N-labeled MpCP3 (left panels) and 5 (right panels), both proteins at 0.5 mM, without NAG4 775

(black) and titrated with 0.1 mM (red), 2.5 mM (green), 5 mM (purple) and 10 mM (pink) of NAG4, 776

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35

highlighting some of the shifted and diminished residue peaks. All spectra were acquired with the same 777

number of scans. (B) Curve fitting of the NAG4 concentration plotted against the ∆δ of specified residues 778

used for the Kd calculation. (C) Computational blind docking models for the interactions between MpCP3 779

and MpCP5 with NAG4. NAG4 is represented by sticks and NAG4-interacting residues are represented as 780

spheres. (D) Relative mRNA expression levels of defense-related tobacco genes after treatment with NAG6 781

(400 nM) or MpCP5 (40 uM) pre-incubated NAG6. 782

FIGURE 5. Sugar hydrolysis and cacao cell wall/chitin binding assays. (A) Colorimetric assay, with the 783

substrate 3,5-dinitrosalicylic acid (DNS), evaluating the glycolytic activity of the recombinant MpCP1, 2, 3 784

and 5 against different substrates. No significant activity was detected for any MpCP analyzed, when 785

compared to positive control TPE (termite total protein extract). (B) Similarly, no relevant interaction 786

between MpCPs and cocoa extracted cell-wall (CCW) was detected in a pull-down assay, when compared 787

to positive control CBM3 (family 3 Carbohydrate Binding Module), from B. subtillis. (C) All MpCPs 788

(soluble form) tested were pulled-down by chitin insoluble polymers, with MpCP3 showing the highest 789

levels of interaction. Chitin is one of the main components of fungal cell wall. S, supernatant fraction; P, 790

pellet fraction. 791

FIGURE 6. Evaluation of self-aggregation capability. (A) Detection of the fluorescence signal induced 792

upon the binding of Thioflavin T (ThT) against increasing concentrations of MpCPs incubated at 37oC, low 793

pH, for 30 days. The data allowed the determination of an amyloid-like self-aggregation behavior for 794

MpCPs 2, 3 and 5, at threshold concentrations between 0.4 and 0.8 mM protein. MpCP1 displays the same 795

behavior, although only at concentrations beyond 1.5 mM, similarly to CpCP (Pazzagli et al. 2009). (B) 796

The signal measured upon the binding of the dyes 1,8-ANS and bis-ANS to MpCP1, 2, 3 and 5 indicating 797

the hydrophobic nature of the aggregates. (C) Time course analysis of the self-assembly of 0.8 mM MpCP2 798

and 3, at 37 oC and low pH, as monitored by ThT fluorescence, shows a much quicker dynamics of 799

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aggregation for MpCP2, which reaches equilibrium after only 50 hours, compared to 25 days required for 800

MpCP3. All the experiments were performed in triplicate. 801

FIGURE 7. Biophysical characterization of the aggregates. (A) Far-UV circular dichroism allowed the 802

tracking of the changes in secondary structure of the MpCPs required for self-assembling (at 0.8 mM 803

concentration). Upon incubation at low pH and high temperature, MpCP1 assumes a completely unfolded 804

structure (with a negative band at 200 nm), while MpCPs 2, 3 and 5 acquire a closer to zero band at 200 nm 805

and more negative ellipticity at 222 nm. No difference was observed in the CD spectra for the MpCPs, 806

between native initial state (0 days) and after 20 days of incubation at 4 °C (data not shown). (B) 807

MRE[θ]200 x MRE[θ]222 combination extracted from the 4°C pH 7.2, and 37°C at low pH spectra for all 808

proteins. The definition of native, pre-molten globule and molten globule regarding to the combined mean 809

residue ellipticity at 200 and 222 nm was taken as revised (Uversky and Fink 2004). (C) Dynamic light 810

scattering data showing the aggregation process as defined by a shift in the Stokes radius after 37°C, low 811

pH incubation. Theoretical values for Rs, calculated using Hydropro (Ortega et al 2011), are 1.83, 1.76, 812

1.73, and 1.95 nm, for monomeric MpCP1, 2, 3 and 5, respectively. (D) Micrographs 1 and 2, obtained by 813

negative stain electron microscopy, depict the filamentous nature of MpCP2 aggregates (0.8 mM, 20 days 814

of incubation at low pH). (E) Cellulose loosening activity was detected for aggregated MpCP2, but not its 815

soluble monomeric counterpart. (F) Aggregated MpCP2 also promotes M perniciosa`s basidiospores 816

germination tube growth. Aggregated n = 12; Soluble n = 37; Buffer n = 34. 817

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1

SUPPLEMENTAL MATERIAL 1

2

Functional diversification of cerato-platanins in 3

Moniliophthora perniciosa as seen by differential 4

expression and protein function specialization 5

6

Mario R. de O. Barsottini a,b**

, Juliana F. de Oliveirab**, Douglas Adamoski

b**, Paulo J. P. L. 7

Teixeiraa, Paula F. V. do Prado

a,b, Henrique O. Tiezzi

b, Mauricio L. Sforça

b, Alexandre 8

Cassagoc, Rodrigo V. Portugal

c, Paulo S. L. de Oliveira

b, Ana C. de M. Zeri

b, Sandra M. G. 9

Diasb**, Gonçalo A. G. Pereira

a,b and Andre L. B. Ambrosio

b** 10

11

aFrom the Departamento de Genética e Evolução, UNICAMP, Campinas, SP 13083-970 12 bLaboratório Nacional de Biociências, CNPEM, Campinas, SP 13083-100 13

cLaboratório Nacional de Nanotecnologia, CNPEM, Campinas, SP 13083-100 14

15

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2

SM Text 16

Experimental Procedures 17

Phylogenetic reconstruction. The phylogenetic tree was estimated with protein sequences 18

aligned with MUSCLE (Edgar 2004). The model of amino acid substitution (WAG+G) was 19

chosen with ProtTest v3.0 (Darriba et al. 2011) and defined in MrBayes v3.2 (Ronquist and 20

Huelsenbeck 2003). The 148.000.000 generations were evaluated in TRACER v1.5 and 21

AWTY (Nylander et al. 2008) to burn-in 103.600.000 (70%) generations (after visual 22

inspections of LnL and cumulative splits plot of putative stationary phase, as well as 23

Effective Sample Size of the last 30% of the chain). The tree was assembled with SumTrees 24

from DendroPy package (Sukumaran and Holder 2010). The protein sequence IDs from the 25

previously described MpCP1 to MpCP5 (Zaparoli et al 2009) are EU250339 (MpCP1), 26

EU250340 (MpCP2), EU250342 (MpCP3), EU250344 (MpCP4) and EU250345 27

(MpCP5). The other sequences from M. perniciosa biotype C and biotype S, M. roreri and 28

C. campanella may be found in the main text. Protein sequences from other fungi were 29

obtained from NCBI and Mycocosm portal (DOE Joint Genome Institute) databases, and 30

their accession numbers are as follows. NCBI accession numbers: AAB0010140 (19 kDa 31

Antigen from Coccidioides posadasii), AAT1191162 (Aca1 from Taiwanofungus 32

camphoratus), EFE4297541 (Asp f13 from Trichophyton verrucosum), EAU3799938 (Asp 33

f15 from Aspergillus terreus), EDN3009454 (BC1G_02163 from Botryotinia fuckeliana), 34

ABM6351348 (Cerato-platanin from Ceratocystis platani), EIW85572 (Cerato-platanin 35

from Coniophora puteana), EDR1116852 (Cerato-platanin from Laccaria bicolor), 36

ACA1360043 (Ceratovariosporin from Ceratocystis variospora), ABE7369260 (Epl1 from 37

Hypocrea atroviridis), CAL8075357 (Epl1 from Trichoderma asperellum), EJD54183 38

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3

(Heat-stable 19 kDa antigen from Auricularia delicata), ABM6350642 (Pop1 from 39

Ceratocystis populicola), EAA5865739 (Predicted protein from Aspergillus nidulans), 40

CCD4351765 (Predicted protein from Botryotinia fuckeliana), EAU8647650 (Predicted 41

protein from Coprinopsis cinerea), EKM58222 (Predicted protein from Phanerochaete 42

carnosa), EED7983453 (Predicted protein from Postia placenta), XP_003032879 43

(Predicted protein from Schizophyllum commune), EGN96314 (Predicted protein from 44

Serpula lacrymans), AAZ8038858 (Sm1 from Hypocrea virens), AAV8379355 (Snodprot1 45

from Gibberella pulicaris), XM0037164 (Snodprot1 from Magnaporthe oryzae), 46

CAC2858561 (Snodprot1 from Neurospora crassa), AAC2687063 (Snodprot1 from 47

Phaeosphaeria nodorum), EEY2380156 (Snodprot1 from Verticillium alboatrum), 48

EAU8063649 (Snodprot1 A from Coprinopsis cinerea), EAU8196951 (Snodprot1 B from 49

Coprinopsis cinerea) and AAM3313059 (Sp1 from Leptosphaeria maculans). Mycocosm 50

protein IDs: 159980 (Predicted protein from Amanita muscaria), 34748 (Predicted protein 51

from Bjerkandera adusta), 266297 (Predicted protein from Gymnopus luxurians), 171081 52

(Predicted protein from Paxillus involutus), 22402 (Predicted protein from Pisolithus 53

microcarpus), 996983 (Predicted protein from Pisolithus tinctorius), 154741 (Predicted 54

protein A from Pisolithus tinctorius), 1069722 (Predicted protein from Pleurotus 55

ostreatus), 102212 (Predicted protein A from Pleurotus ostreatus), 45615 (Predicted 56

protein B from Pleurotus ostreatus), 1110827 (Predicted protein C from Pleurotus 57

ostreatus), 13911 (Predicted protein A from Serpula lacrymans), 61724 (Predicted protein 58

from Sphaerobolus stellatus), 506951 (Predicted protein from Suillus luteus), 73763 59

(Predicted protein A from Suillus luteus). 60

61

62

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4

Crystallization, X-ray Crystallography Data Collection and Processing. Crystallization 63

experiments were performed at 18oC using the conventional sitting drop vapor diffusion 64

technique. Drops were made by mixing equal parts of protein (at 10, 40, 20 and 40 mg/mL 65

for MpCP1, MpCP2, MpCP3 and MpCP5, respectively) and well solution. The 66

crystallization conditions are described in the main text. Before data collection at cryogenic 67

temperature, MpCP3 harvested crystal was cryoprotected with 10% ethylene glycol added 68

to the mother liquor, while MpCP1’s, MpCP2’ s and MpCP5’s crystal were collected 69

without additional cryo-protection. 70

71

Structure Solution and Refinement. The structure of MpCP1 was solved by Sulfur-SAD. 72

The redundant anomalous data allowed the unambiguous identification and phasing of 7 73

sulfur atoms using SHELXC/D/E (Sheldrick 2010). Using ARP/wARP (Perrakis et al 74

1999) it was possible to build 116 out of the 127 residues expected for the monomer. 75

Positional and B-factor refinement cycles were then made with Refmac (Vagin et al. 2004). 76

Manual building of the extra portions and real space refinement, including Fourier electron 77

density map inspection, were performed with Coot (Emsley et al. 2010). Solvent water 78

molecules, treated as oxygen atoms, were added using the appropriate Coot routine. The 79

first set of phases of MpCP2 and MpCP3 was obtained by the molecular replacement 80

technique as implemented in the program MrBUMP (Keegan and Winn 2008), using the 81

MpCP1 monomer (deposited in PDB ID: 3SUJ) as the search model. Real space and 82

reciprocal space refinements were performed with Coot and Refmac, respectively. The set 83

of phases of MpCP5 was obtained by the Phaser program (McCoy et al. 2007) using the 84

protein Sm1 from Trichoderma virens as the search model (deposited in PDB ID: 3M3G). 85

Following molecular replacement, density modification was performed using the program 86

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5

Parrot, which is part of the CCP4 suite (Winn et al 2011) and the improved map then 87

submitted to automated interpretation by the ARPwARP routine. Positional and B factor 88

refinement cycles were performed with Refmac. For calculation of Cα r.m.s.d values we 89

used the algorithm SSM (secondary structure matching) as implemented in Coot. 90

91

NMR sample preparation. To prepare 15N- and 13C/15N-labelled MpCP proteins, the E. 92

coli strain SHuffle (NEB) was cultivated in M9 minimal media supplemented with 1 g/L 93

15NH4Cl (Cambridge Isotope Laboratories) combined with 4 g/L D(+)-glucose (Sigma) to 94

obtain only 15N-labelled proteins or with [13C6]-D(+)-glucose (Cambridge Isotope 95

Laboratories) to obtain double-labeled proteins. The cultures were grown at 30°C until 600 96

nm absorbance of 1.0 followed by the protein expression induction with 0.5 mM isopropyl-97

β-D-thiogalactoside (IPTG) for further 14 hours at 30°C. The purification protocol was 98

performed essentially as described for the unlabelled protein. 99

100

N-acetylglucosamine titration NMR experiments. Titrations of 15N-labeled MpCP 101

proteins with the carbohydrate N-acetylglucosamine (NAG, Sigma-Aldrich, A8625) in its 102

tetrameric form (NAG4, Megazyme O-CHI4) were followed by the recording of the two-103

dimensional 15N-HSQC spectra. The titration series was performed by direct addition of 104

small aliquots (0.3 to 30 µL) of the carbohydrate (200 mM stock) to 600 µL of 15N-MpCPs 105

(50 uM) in order to obtain carbohydrate final concentrations of 0.1, 2.5, 5 and 10 mM. The 106

monomeric NAG was added to a final concentration of 10 mM. The temperature was 107

maintained at 25°C throughout the NMR experiment. The carbohydrates binding constant 108

was determined by spectral changes in the protein 15N-HSQC chemical shift values as a 109

function of the concentration of unlabeled ligand. The chemical shift perturbation of the 110

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6

15N-HSQC spectra was normalized according to the equation ∆δ(15N+1H) = 111

[(∆δ15N/10)2+(∆δ1H)2]1/2 (ppm) (Cavanagh el al. 2007). The dissociation constant values 112

(Kd) were calculated based on chemical shift changes of eight residues of each MpCP 113

(Phe32-Asp39 for MpCP3 and Val108-Lys115 for MpCP5), using the program xcrvfit. 114

115

N-acetylglucosamine docking to MpCP3 and MpCP5, mutagenesis and fluorescence-116

based thermal shift assays. NAG4 ligand structure in Mol format was obtained from 117

ChemicalBook website (CAS #2706-65-2). NAG4, MpCP3 and MpCP5 structures were 118

first submitted to energy minimization using YASARA (Krieger et al. 2009). For that, all 119

hydrogen atoms and other missing atoms from the model were created using force field 120

parameters obtained from YAMBER3. All receptor water molecules were removed. A short 121

steepest descent energy minimization was carried until the maximum atom speed dropped 122

below 2200 m/s. Then 500 steps of simulated annealing were performed with a temperature 123

of 0K. A simulation cell was centered upon the MpCPs structures and its extension defined 124

as the extreme coordinates of protein plus twice the size of the ligand radius. The size of the 125

docking grid for MpCP3 and MpCP5 complexes were 86 Å × 79 Å × 75 Å and 95 Å × 80 126

Å × 77 Å, respectively. Grid spacing used for MpCP3 and MpCP5 complexes were 0.685 127

and 0.766 Å, respectively. All docking simulations were performed by AutoDock 4.2.3 128

called from YASARA. The number of Lamarckian genetic algorithm (LGA) runs was 100 129

for each molecular complex. Population size was 150 individuals and 15 random torsions 130

for each individual. The 100 independent LGA runs were processed using the built-in 131

clustering analysis as implemented in YASARA with a 5.0 Å cutoff. 132

Mutants were obtained with the QuikChange Site-Directed Mutagenesis Kit 133

(Stratagene) following the manufacturer’s instructions and purified according to the 134

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7

protocol described for the wild-type proteins. To evaluate the ability of wild type and 135

mutants MpCPs 3 and 5 to bind to NAG4 we performed thermal shift assays in a 7500 Real 136

Time PCR System (Applied Biosystems). Fifty micromolar of protein and a 100-fold 137

dilution of a 5000-fold SYBRGreen stock solution (Life Technology) were incubated in the 138

absence and presence of 20 mM or 60 mM of NAG4 (for MpCP3 and MpCP5, 139

respectively), in 50 mM Tris-HCl, pH 8,5 and 150 mM NaCl solution. The fluorescence 140

signal was measured between 20°C and 100°C with a 1°C step per cycle. A sigmoidal 141

curve was fitted to the data points between 50oC and 80oC (90oC for MpCP5) using Origin 142

8.0 (OriginLab Corp., Northamptom, NA) and the inflection point taken as the melting 143

temperature (Tm) of the proteins. 144

145

qPCR. Three-week-old tobacco seedlings were thoroughly washed with distilled water, had 146

their roots removed with a scalpel and were left in distilled water for 5 h in order to recover 147

from the injury. Each seedling (approx. 100 mg of plant material) was transferred to plate 148

dish containing 300 µL of either 20 mM Na2HPO4-NaH2PO4 pH 7.2 solution, 400 nM of 6-149

acetylglucosamine (NAG6, Megazyme O-CHI6), or MpCP5 (40 µM) pre-incubated with 150

NAG6 (400 nM) for 24 hours. After 24 h, the seedlings were flash frozen in liquid nitrogen 151

and stored at -80 °C. RNA extraction was performed with RNeasy Plus Mini Kit (Qiagen) 152

following the manufacturer’s instructions. cDNA synthesis was performed with Superscript 153

III First-Strand Synthesis System for RT-PCR (Invitrogen), following the manufactures`s 154

instructions and the random hexamers primers. qPCR was performed in triplicate with 155

Power SYBR Green PCR Master Mix (Applied Biosystems) and the equipment ViiA7 156

Termocycler (Applied Biosystems). Data was evaluated following the 2-∆∆Ct (Livak and 157

Schmittgen 2001). Primers used for gene expression quantification were: 5’-158

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CCGTTGCTGCGATGATTCA-3’, 5’-GCTGCCTTCCTTGGATGTG-3’ (rRNA 18S; 159

Genbank accession number AJ236016.1), 5’-GACATCAGCGGAAGCAGTAGTAT-3’, 160

5’-CTTAGCGTCGGCGAAGTAGT-3’ (npr1; DQ837218.1), 5’-161

TGCTCTCCGAACATCTCCACAATG-3’, 5’-CCGATAGGAGTGCCTTGGAAGTTG-3’ 162

(pal1; M84466.1), 5’-GATGCCGACAAGCCTCTC-3’, 5’-163

TTGAGTTCCTGTTCCTGTGTTC-3’ (pr4a; X60281.1), 5’-164

GCTGTTACTCATGCTGCCACTT-3’ and 5’-AATGCGAGCCTGGACTGTTC-3’ (pr5; 165

X15224.1). 166

167

Cacao cell wall extraction. Extraction of cacao leaves cell wall was performed as 168

described elsewhere (Melton and Smith 2001) with minor modifications. Theobroma cacao 169

var. Comum was cultivated in a greenhouse under controlled temperature and humidity 170

conditions (from 22 to 28 °C and above 50%, respectively). Fifteen grams of healthy fully-171

expanded leaves from three months old plants were extirpated from their central nerves and 172

grinded with a mortar and pestle in the presence of liquid nitrogen. The obtained powder 173

was transferred to 300 mL of 50 mM Hepes pH 7.0 and 10 mM Dithiothreitol (DTT) and 174

quickly homogenized with a blender. The suspension was filtered through a filter paper and 175

extensively washed with 50 mM Hepes pH 7.0 followed by distilled water. The retentante 176

was lyophilized and stored at -80°C. 177

178

Glycolytic activity. MpCPs glycolytic activity was measured using a colorimetric assay 179

with the substrate 3,5-dinitrosalicylic acid (DNS) (Miller 1959), with fifty micrograms of 180

each MpCP and the positive control termite total protein extract (kindly donated by Dr. 181

Fábio Márcio Squina, CTBE, CNPEM, Campinas, SP, Brazil), and five hundred 182

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9

micrograms of various cell-wall polyssacharides. were incubated with 0.5% (w:v) of either 183

carboxymethyl cellulose, arabinan, arabinoxylan, laminarin, xylan, pectin, chitosan, 184

arabinogalactan, manan, starch, or CCW in 50 mM citrate buffer pH 5.0 at 37°C for 1h, 185

after which it was added to one volume of DNS solution (1% DNS, 0.2% phenol, 0.05% 186

Na2SO3 and 1% NaOH). The samples were heated at 95 °C during five minutes and the 540 187

nm absorbance measured in a 96-well plate (Eppendorf) with the Infinite 200 PRO Plate 188

Reader (Tecan). All reactions were made in triplicate. 189

190

Pull-down assays. The cocoa cell-wall extract (CCW) or chitin from shrimp shell (Sigma-191

Aldrich, C7170) interaction with MpCP1, MpCP2, MpCP3, MpCP5 (either soluble or 192

aggregated protein) and the positive control purified recombinant CBM3 (family 3 193

carbohydrate-binding module) from Bacillus subtillis (kindly donated by Dr. Mario 194

Murakami, LNBio, CNPEM, Campinas, SP, Brazil) was assessed by pull-down assay. Fifty 195

micrograms of each protein were incubated with either 20 mg of CCW or 5 mg of chitin in 196

a final volume of 1 mL binding solution (50 mM Tris-HCl pH 8,5, 150 mM NaCl). After 197

overnight incubation at room temperature, the insoluble fraction was separated by 198

centrifugation, the pellet washed three times with binding solution and then subjected to 199

SDS-PAGE 15 % analysis. 200

201

Thioflavin T, 1,8-ANS and bis-ANS fluorescence measurements. To study MpCP1, 202

MpCP2, MpCP3 and MpCP5 capacity of forming ordered amyloid-like aggregates we 203

incubated 0.1, 0.2, 0.4, 0.8, 1.5 and 3.0 mM of protein in 20 mM Na2HPO4-NaH2PO4 pH 204

7.2, 10% acetic acid solution (final pH of 3) for up to 30 days at 37°C as described 205

previously (Pazzagli et al. 2009). As controls, the proteins were incubated in 20 mM 206

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Na2HPO4-NaH2PO4 pH 7.2 at 4°C and 37°C. MpCPs ordered aggregates formation was 207

monitored by fluorescence spectroscopy using Thioflavin T (ThT; Sigma-Aldrich) probe. 208

ThT is known to fluoresce upon contact with amyloid-like aggregates (Biancalana and 209

Koide 2010, Khurana et al. 2005, Marcon et al. 2005). Beyond 0.8 mM of MpCP2, 3 and 5, 210

the protein solution became jellified, thus precluding sample handling and analysis. A fresh 211

0.22 uM filtered stock solution of 1 mg/mL of ThT in 50 mM Glycine-NaOH pH 8.5 was 212

prepared before each measurement. The stock concentration was double checked by 213

absorbance measurement at 475 nm in a Nanodrop 2000C spectrometer (Thermo Scientific) 214

and comparison with a calibration curve, and then diluted to 10 µM in 50 mM Glycine-215

NaOH pH 8.5 before use. Two nanomols of protein were diluted to 20 µL in 50 mM 216

Glycine-NaOH pH 8.5 solution and centrifuged at 20.000xg for 10 minutes. The 217

supernatant was removed and 200 µL of 10 µM ThT solution was added to the pellet (final 218

protein concentration of 10 µM considering complete protein precipitation). The sample 219

was then vortexed for one minute and the fluorescence measurements recorded (excitation 220

wavelength of 435 nm and emission wavelength of 485) on a plate reader 221

spectrofluorimeter (EnVision, Perkin Elmer) using a 96-well all-black-walled plate 222

(Eppendorf) and the default configurations of the equipment at 25oC. Time course of 223

aggregation of MpCP2 and 3 at 0.8 mM was monitored using ThT fluorescence during 3 224

and 30 days, respectively. 225

1,8-ANS and bis-ANS measurements were made accordingly to previously 226

published (Pazzagli et al. 2009), using MpCPs (at 0.8 mM) incubated at 37°C in low pH 227

solution for 5 and 20 days or control proteins incubated at 4°C in 20 mM Na2HPO4-228

NaH2PO4 pH 7.2 for 20 days. The final protein concentration used in the measurements was 229

2 µM, with 10 µM of ANS or 2 µM of bis-ANS. 230

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231

Cellulose fragmentation. Fragmentation activity of soluble and aggregated MpCP2 on 232

cellulose was assayed as described elsewhere (Baccelli et al. 2013). Briefly, 0.5 mm 233

diameter whatman n. 1 filter paper disks were incubated at 37 °C in a 96-well plate with 40 234

μM of protein or 20 mM Na2HPO4-NaH2PO4 pH 7.2 solution to a final volume of 100 uL. 235

The pH of the aggregated MpCPs solution was adjusted to 7.2 prior to the experiment. 236

After 72 h, 500 nm absorbance of the supernatant were measured in a plate reader 237

(EnVision; Perkin Elmer). 238

239

Spore germination. Spores obtained as described in the main text (RNA-seq methodology) 240

were gently centrifuged and ressuspended in distilled water and then added to soluble, or 241

aggregated (pH adjusted to 7.2) protein samples (40 μM), as well as 20 mM Na2HPO4-242

NaH2PO4 pH 7.2 and incubated in the dark at 28°C for 6 h. Samples were inspected in an 243

optical microscope for germination rate and germination tube length measurements by 244

using the ImageJ software. 245

246

Circular dichroism spectroscopy. Eight hundred micromolar of protein were incubated at 247

37°C in 20 mM Na2HPO4-NaH2PO4 pH 7.2 or in a low pH solution containing 20 mM 248

Na2HPO4-NaH2PO4 pH 7. 2 and 10% acetic acid for up to 30 days and then diluted to 10 249

µM in Milli-Q water for immediate measurement by far-UV CD spectroscopy (190-260 250

nm) using a JASCO J810 spectropolarimeter. The CD spectra were acquired at 20°C in a 251

0.1 cm optical path cuvette with a band-width of 1 nm, response time of 4 s, data pitch of 252

0.5 nm and scanning speed of 100 nm/min. Each data point was generated by averaging ten 253

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accumulations, subtracted from the blank spectrum and converted to mean residue molar 254

ellipticity (MRE [θ]). 255

256

Identification of native MpCPs by mass spectrometry. M. perniciosa FA553 was 257

cultivated in light malt extract culture medium (0.17% malt extract, 0.5% yeast extract) 258

complemented with 5% glycerol under agitation at 25°C. After seven days of growth, the 259

hyphae were removed by filtration through a nylon mesh and the culture medium dialyzed 260

against three changes of distilled water (Spectral/Por, Spectral Laboratories, Inc.; nominal 261

cutoff of membrane of 3.5 kDa). The dialyzed media was filtered through a 0.22 µM PVDF 262

syringe filter (Millipore) and concentrated 100-fold with a Amicon Ultra filter device 263

(Millipore) (MWCO of 10 kDa). The final sample was subjected to Comassie Blue-stained 264

15% SDS-PAGE analysis. The gel lanes were cut into small slices, the protein digested 265

with trypsin (Promega), extracted from the gel and desalted with the trapping column 266

Symmetry C18 (180 µm x 20mm, Waters) at a flow rate of 5 µl/min for 2 min. LC/MS-MS 267

analysis was performed with a C18 1.7 µm BEH 130 (100 µm x 100 mm) RP-UPLC 268

(nanoAcquity UPLC, Waters) chromatography coupled with nano-electrospray tandem 269

mass spectrometry on a Q-Tof Premier API mass spectrometer (MicroMass/Waters) over a 270

0-50% acetonitrile gradient in 0.1% formic acid for 45 min (flow rate of 600 nL/min). The 271

instrument was operated in MS positive mode, with data continuum acquisition from m/z 272

100–2,000Da at a scan rate of 1 s and an interscan delay of 0.1 s. The identification of the 273

peptides detected from LC/MS-MS experiments were done with the Mascot Distiller 274

v.2.3.2.0 (Matrix Science, Boston, MA) program using carbamidomethyl–cys as fixed 275

modification (monoisotopic mass 57.0215Da), methionine oxidation as variable 276

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modification (monoisotopic mass 15.9949), 0.1Da MS and MSMS fragment tolerances and 277

using the M. perniciosa database (17,105 sequences). 278

279

Transmission Electron Microscopy. Transmission Electron Microscopy was performed 280

with MpCP2 (0,8 mM) after 20 days of incubation at low pH. For visualization by negative 281

stain, protein samples at a concentration of 1.0 mg/mL were prepared in Tris-HCl buffer 282

(30 mM Tris-HCl pH 8.5, 50 mM NaCl). Holey carbon-coated grids were glow discharged 283

for 25 seconds at 15 mA using an easiGlow system (PELCO). A 3 µL sample was 284

deposited onto the grid for 60 seconds followed by two steps of blotting and staining with 3 285

µL of 2% uranyl acetate (30 seconds). Images were recorded at -3 µm defocus at x50,000 286

magnification using a Jeol JEM-2100 operating at 200 kV. Images were taken using an F-287

416 CMOS camera (TVIPS). 288

289

Studies of protein cleavage associated to self-assembling by mass spectrometry. Protein 290

cleavage studies were performed as decribed elsewhere (Pazzagli et al. 2009). Eight 291

hundred micromolar of MpCP1, 2, 3 and 5 were incubated in low pH solution for 20 days 292

for the self-assembly formation as confirmed by ThT fluorescence. Two hundred 293

micrograms (20 µL) were then fractionated with MicroconYM30 (cut-off of 30 kDa; 294

Milipore) and the flow through collected (FT). The Microcon was then washed with water 295

twice and the retentate dissolved in 20 µL of 0.1% TFA after vigorous vortexation 296

(retentate fraction). Both retentate and FT fractions were assayed for ThT fluorescence. 297

Half of each fraction was treated with 10 mM dithiothreitol for 25 min at 56°C and then 298

with 50 mM iodoacetamide during 30 min at room temperature for disulfide bridge 299

disruption. Samples of retentate fractions were also digested with tripsin (Promega) during 300

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16 hours at 37°C. All the fractions were dried in speed vacuum, dissolved in 0.1% formic 301

acid and submitted to LC/MS-MS analysis as described above, with data continuum 302

acquisition from m/z 100–3,000Da. 303

304

Injection of MpCPs in tobacco leaves. MpCP1, 2, 3 and 5, at a final concentration of 0.5 305

mg/mL (approximately 40 uM) in 20 mM Na2HPO4-NaH2PO4 pH 7.2 were individually 306

infiltrated into the central nerves leaves of Nicotiana tabacum var. Petite Havana (4-6 307

weeks old). Right after the infiltration, the leaves were detached and left inside of a dark 308

moist chamber for 24 hours, upon which they were inspected for necrotic symptom and 309

phytoalexin production. Phytoalexin production was measured by the auto-fluorescence 310

property of the phytoalexin compounds and registred by the Gel Logic 2200 Imaging 311

System (Carestream) (excitation at 365 nm and emission at 535 nm). 312

313

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414

Author-Recommended Internet Resources 415

416

http://tree.bio.ed.ac.uk/software/tracer/ 417

http://www.bionmr.ualberta.ca/bds/software/xcrvfit/html-v4.0.12/index418

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SM Tables 419

Supplemental Table 1A: Peptide sequences identified by LC/MS-MS mass spectrometry of 420

cultivating media of M. perniciosa in vitro- grown necrotrophic mycelium. 421

Supplemental Table 1B: Sequence alignment between the CpCP, MpCPs 1-12 and Sm1 422

highlighting in yellow the N-X-S/T sequence predicted to be glycosylated by the NetNGlyc 423

1.0 online server. NGS sequence on Sm1 was experimentally shown to be glycosylated 424

(Vargas et al. 2008). The colors follow the color pattern stipulated for the clusters Mper1 425

(blue), Mper2 (green) e Epl1/Epl2 (light red) on Figure 2 of the main text. 426

Supplemental Table 2: X-ray crystallography data collection parameters and structure 427

refinement statistics. 428

Supplemental Table 3: Peptides identified by mass spectrometry of MpCP1, 2, 3 and 5 flow 429

through fraction after low pH-induced aggregation and fractionation. 430

431

SM Figures Legends 432

Supplemental Figure S1: MpCP proteins sequence alignment. ClustalW (Larkin et al. 2007) 433

was used to align all identified CPs from M. perniciosa as well as the CP from C. platani 434

(CpCP). In green, residues 100% conserved throughout all sequences. In cyan, MpCP3 435

residues involved on NAG4 binding (as shown by NMR and docking, and confirmed by 436

mutagenesis), as well as equivalent ones found conserved in other sequences. Equivalent 437

residues in CpCP are also among those mostly disturbed upon NAG4 binding (de Oliveira 438

et al 2011). The pairs of cysteines involved in disulfide bridges, which are also fully 439

conserved across the aligned sequences, are depicted in yellow and are connected by 440

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straight lines. The NAG4-binding regions in MpCP3, as judged by NMR and the docking 441

model (loops between β2 and β3 and helices α1 and α2) are surrounded by red boxes 442

Supplemental Figure S2: Crystal structure of MpCP1 highlighting the coordinating ions. 443

Cartoon representation of the polypeptide chain. Five strong peaks (over 7σ in height) were 444

observed in the Fourier difference electron density map for MpCP1, which, with respect to 445

their contributions to the dispersive component of the scattering factor, were interpreted as 446

three zinc ions (lying in the proximity of residues Asp69, Glu106 and Asp137), one sodium 447

ion (coordinating with Glu124) and a chloride ion (coordinating with the zinc ion near 448

Asp69). The ions of zinc, chloride and sodium are depicted as grey, green and purple 449

spheres, respectively. Respective coordinating residues are shown as sticks. Omit Fourier 450

difference map (at a sigma level of 3, green mesh), were calculated from the final 451

coordinate file, excluding the ions. 452

Supplemental Figure S3: Binding of NAG and NAG4 to MpCPs as detected by NMR 453

spectroscopy. Overlay of the 15N-HSQC spectra of 15N-labeled MpCP1, 2, 3 and 5 (all 454

proteins at 0.05 mM) without ligand (black) and with 10 mM (pink) of NAG (A) or titrated 455

with 0.1 mM (red), 2.5 mM (green), 5 mM (purple) and 10 mM (pink) of NAG4 (B). All 456

spectra were acquired with the same number of scans. When a protein and its ligand 457

interact, the chemical environment of the backbone amides changes and this usually causes 458

a change in chemical shift. Such changes can be grouped into three regimes: fast, 459

intermediate, and slow. The regime observed for any amide depends on the relationship 460

between the chemical shift measurement and the rate of exchange between the free and 461

bound states. If the exchange rate were higher than the chemical shift, a single peak would 462

appear at a position between the chemical shifts of the free and bound forms. If the 463

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exchange rate is lower than the chemical shift, two peaks would be observed, one for the 464

free form and one for the bound. In the intermediate case (the exchange rate is comparable 465

to the chemical shift difference), the peaks become broadened and may be unobservable 466

(Mott et al. 2003). During the titration of MpCP3 with NAG4, we could only observe peak 467

shifting phenomena, a clear indication that some residues of this complex are in the fast 468

exchange limit. On the MpCP5-NAG4 spectra, however, we could observe that, in addition 469

to the peak shifting behavior, the intensities of some peaks gradually decrease indicating 470

that the off-rate of the complex is in intermediate exchange on the NMR time scale. Since 471

the binding constant could not be calculated from the intensity data of the residues in 472

intermediate exchange because their peak intensities dropped sharply when the NAG4 473

concentration increased, the affinity estimated from residues in fast exchange may be 474

underestimate. (C) Scaled chemical shift differences between the 15N-HSQC spectra of the 475

proteins without NAG4 and incubated with 10 mM of NAG4, with affected residues 476

labeled. A cut-off of 0.05 ppm was considered. Scaling was performed according to the 477

equation ∆δ(15N + 1H) = [(∆δ15N/10)2 + (∆δ1H)2]1/2 (upper panel). MpCP5-NAG4 chemical 478

shift graphic depicting the plot of fractional changes in peak intensities for individual 479

backbone amide resonances of MpCP5 upon addition of NAG4. The axis Y presents the 480

relative peak intensity change (I-I0)/I0 of each assigned cross-peak caused by the addition of 481

NAG4, where I is the peak intensity measured in the presence of 10 mM NAG4 and I0 is 482

the peak intensity measured in the free protein. All the residues under either a fast or 483

intermediate off-rate regime are listed on the right followed by the chemical shift difference 484

value (middle panel) or relative peak intensity (lower panel). (D) Surface representation of 485

MpCP5 crystal structure highlighting the regions likely involved in NAG4 binding (in blue, 486

residues presenting chemical shift difference and in red residues displaying chemical shift 487

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broadening upon NAG4 addition). (E) ClustalW (Larkin et al. 2007) alignment of MpCP4, 488

5 and 11 showing the conservation of the MpCP5 residues which peaks displayed chemical 489

shift difference (blue) and signal suppression (red) upon NAG4 binding. The MpCP5 490

regions affected by the NAG4 binding involve residues present in β-strand 1 and α-helix 1, 491

as well as the connecting loop (Leu24, Iso25, Lys26, Trp27, Asp28, Asp29, Lys30 and 492

Phe31), the β-hairpin between β-strands 3 and 4 (Tyr88, Lys89, Gly90), part of β-strand 4 493

(Asn91, His92), the loop connecting β4 and β5 (Arg100), part of β5 (Val101, Glu102, 494

Glu103), the loop connecting β5 and α4 (Val108, Gly109 and Gly110), α-helix α4 (Thr111, 495

Asp112, Val114, Lys115, Asn116, Leu117, Thr118), the loop connecting α4 and α5 496

(Thr119, Phe120, Ala123), part of α-helix α5, the connecting loop between α5 and β6 and 497

the β-strand β6 (Tyr127, Trp129, Gly130, Thr131, Ala132, Gln133, Leu134). 498

Supplemental Figure S4: Fluorescence-based thermal shift assay showing MpCP3 and 5 499

residues involved in NAG4 binding. NAG4 binding to MpCP3 wild-type leads to an 500

increase on protein stability (higher melting temperature, Tm). Mutated MpCP3 protein 501

(Ala36Thr/Asp39Gly and Trp68Tyr) are less stable in the presence of NAG4 (left panel). 502

MpCP5 mutations Thr119Ser and Thr119Ala did not disturbed NAG4-induced thermal 503

stability, while Asn116Lys mutation apparently increased protein affinity for NAG4, as 504

judged by an increase on Tm upon NAG4 binding (right panel). These results indicate that 505

the MpCP5:NAG4 binding surface is rather large and likely involves the simultaneous 506

interaction of more than one molecule. 507

Supplemental Figure S5: Structure of MpCP1 and comparison with other proteins that 508

present a double Ψβ-barrel fold and interact with polysaccharides. (A) Cartoon 509

representation of MpCP1 (red) and E. coli expansin EXLX1 (Kerff et al. 2008) (gray; PDB 510

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ID: 3D30) superimposed. (B) Cartoon representation of MpCP1 and GH45 endoglucanase 511

MeCel45A (unpublished data) (PDB ID: 1WC2) superimposed. (C) Cartoon representation 512

of MpCP1 and EcMltA(D308A)–chitohexaose (van Straaten et al. 2007) (PDB ID: 2PI8) 513

superimposed. Chitohexaose molecule, co-crystallized with EcMltA, is represented by 514

spheres. Superposition using the secondary structure matching (SSM) algorithm (Krissinel 515

and Henrick 2004) was performed with the program Coot (Emsley et al. 2010). 516

Supplemental Figure S6: RMN spectroscopy of MpCP1 and MpCP5 after 4 days of 517

incubation in low pH solution. Overlay of initial (black) and after 4 days of incubation at 518

37oC in low pH (green) 15N-HSQC spectra of 15N-labeled MpCP1 and 5 (at 0.1 and 0.4 519

mM, respectively) indicating MpCP1 (left panel) unfolding tendency (the majority of the 520

resonance peaks in the 15N-HSQC spectra are clustered between 8.0 ppm and 8.8 ppm in 521

the 1H dimension, characteristic of unfolded structures; Cavanagh et al. 2007) and MpCP5 522

(right panel) aggregating behavior (aggregating protein has a tendency of losing intensity 523

signa; Cavanagh et al. 2007). 524

Supplemental Figure S7: Identification of the aggregating peptides by mass spectrometry. 525

The C. platani CP self-aggregation correlates with a fragmentation process as for some 526

amyloid-like proteins which undergo self-assemblage after cleavage of the polypeptide 527

chain (de Laureto et al. 2005, Frare et al. 2004, Mishra et al. 2007, Pazzagli et al. 2009, 528

Tsiroulnikov et al. 2006). Having shown that the MpCPs also undergo self-assembling 529

under mild-denaturing conditions we set out to learn whether this process was governed by 530

protein fragmentation. (A) Comassie stained SDS-PAGE gel of MpCP1, 2, 3 and 5 531

incubated at 4oC and 37oC under pH 7.2 and at 37oC in low pH solution for 10, 15, 20 and 532

25 days. It was evident in the electrophoresis results that after 10 days all the proteins at 533

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low pH had already presented alterations on their migration behavior compatible with a 534

degradation process. In order to identify the fragments, 0.8 mM of MpCP1, 2, 3 and 5 were 535

incubated for 20 days in low pH at 37°C or in pH 7.2 at 4°C and samples were fractionated 536

to separate the aggregated protein from the cleavage products (flow through fraction, FT). 537

(B) MpCPs and CpCP protein sequences aligned with ClustalW (Larkin et al. 2007). 538

Protein segment identified by LC/MS-MS in the flow-through fraction (cleaved-off 539

fragments) are colored according to the structure representation color. Aggregating peptides 540

are underlined. The disulfide bridges are indicated by connecting lines. The FT (which did 541

not present ThT fluorescence, Figure S8) were then analyzed by nanospray LC/MS-MS, 542

with a 3 KDa cut-off for mass fragmentation. FT fraction analysis of protein incubated in 543

pH 7.2 at 4°C did not reveal any peptide (data not shown). On the other hand, the observed 544

fragmentation pattern of the proteins incubated in low pH at 37°C revealed that the major 545

cleavage sites for the isoforms 1, 2, 3 and 5 are in the N-terminus and in the regions 546

peripheral to the two disulphide bridges, in clear accordance with what was described for 547

CpCP (Pazzagli et al. 2009). Several attempts of characterizing the aggregate-forming 548

peptides by LC/MS-MS were made, which included tripsinization of the retentate fraction, 549

but they all proved unsuccessful. By excluding what was identified in the FT fraction, we 550

concluded that MpCP1 presented the shortest aggregating-peptide (Ile70-Asp102), while in 551

the remaining isoforms they are formed by structurally correlated regions (Ser73-Arg91, 552

Ser72-Gly83 and Cys80-Lys89, for MpCP2, 3 and 5, respectively) added to non-553

consecutive segments that are hold together by the two disulphide in MpCP2 and 3. More 554

specifically, MpCP2 aggregating-peptide is covalently joined to helix α2 and part of α6, 555

MpCP3 to α2 (and adjacent loops) and part of α6, MpCP5 to helix α6 and β strands β6 and 556

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β6*. (C) Cartoon representation of the MpCP1 (red), 2 (blue), 3 (yellow) and 5 (green) 557

structures with the aggregating peptides colored in gray. See also Table S3. 558

Supplemental Figure S8: ThT fluorescence of retentate and flow through fractions of 559

MpCP1, 2, 3 or 5 aggregates before and after fractionation. Eight hundred micromolar of 560

each protein were incubated for 20 days in low pH solution and ThT fluorescence measured 561

for the total solution and for the flow through (FT) and retentate fractions, after 562

fractionation with MicroconYM30 (cut-off of 30 kDa, Milipore). Most of the ThT 563

fluorescence detected in the initial protein solution is measured in the retentate fraction, 564

clearly showing that it contains the aggregates. Background refers to ThT fluorescence 565

without the protein. MpCP1 aggregate presents low ThT fluorescence at 0.8 mM protein 566

concentration. 567

Supplemental Figure S9: MpCP1, 2, 3 or 5 injection into tobacco leaves did not induce 568

neither phytoalexin production nor necrosis. MpCP1, 2, 3 and 5 at a final concentration of 569

0.5 mg/mL were individually infiltrated into the central nerves leaves of Nicotiana tabacum 570

var. Petite Havana (4-6 weeks old). Right after the infiltration, the leaves were detached, a 571

picture was taken (left panel, 0 hours) and the leaves were kept inside of a dark moist 572

chamber for 24 hours, upon which they were inspected for phytoalexin production (A) and 573

necrotic symptom (B), having tested negative for both outputs (right panels, 24 hours). 574

575

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Supplemental Table 1 A 576

Peptide sequences identified by LC/MS-MS mass spectrometry of cultivating media of M. perniciosa in vitro- grown necrotrophic 577

mycelium. Mass (exp), experimental mass; Mass (calc), calculated mass. In yellow, segment identified by the NetNGlyc 1.0 as 578

putatively glycosylated (see Supplemental Table 1 B). 579

580 581 582 583

Protein Peptide sequence identified m/z Charge

Mass

(exp),

Da

Mass

(calc),

Da

Number

of times

identified MpCP2 FDNTYDNASGSMNTVACSTGANGLSQR 1419.5608 +2 2837.1070 2837.1981 5

FDNTYDNASGSMNTVACSTGANGLSQR 946.7344 +3 2837.1815 2837.1981 2

SINLVAIDHAGNGFNVAQAAMDELTNGNAVALGTIDVQSQQVAR 1498.4107 +2 4492.2103 4492.2405 5

MpCP3 TIHVVGVDVAGNGFNVGQR 970.0119 +2 1938.0092 1938.0072 1

TIHVVGVDVAGNGFNVGQR 647.0073 +3 1938.0001 1938.0072 8

GYSTFGSVPSYVGAVDTITGWNSESCGTCYQITWSGTGK 1394.2909 +3 4179.8509 4179.8467 4

MpCP7 LQYDPTYDNR 642.7943 +2 1283.5740 1283.5782 8

GFSTFGSLPNFPR 713.8508 +2 1425.6870 1425.7041 12

DGSLGTTACSDGPNGLITK 932.4243 +2 1862.8340 1862.8680 4

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Supplemental Table 1 B

CpCP 1 -----MKFSI------LPMIASAMA------------------VSISYDPIYA-ADLSMGSVACSNGDH--GLMAQ--YPTLGEVPGF-----PNVGGIPDIAGWDSPSCGTCWKVTIPN 81

Sm1 1 -----MQLSNIF--TLALFTAAVSA------------------DTVSYDTGYDNGSRSLNDVSCSDGPN--GLETRYHWSTQGQIPRF-----PYIGGAAAVAGWNSASCGTCWKLQYS- 87

MpCP1 1 -----MKSIAIFTPILILLTISAGA------------------VKLSYDEAYDNPSSSLLSVTCSDGEN--GLYPK--YRTFGDLPGF-----PCIGGSSDIAGYNSPNCGSCYQLTYSS 88

MpCP9 1 -----MKFFALC--STLALVSSTTA------------------VNLAWDDAYNDRAGSLNAVACSDGAN--GLETRFGFAVFGDIPTF-----PFVGGAPNIPGWNSSACGTCWALTFVN 88

MpCP4 1 -----MKSFTTL----LSLGFVASVASALPSPQGDPTPATPAKYRLSHNPVYDNRSMEIGRLACGGGEN--GLVT-KGYRTLGDLPFF-----DNIGAA-HPSSYNSLECGSCWNLTYK- 101

MpCP5 1 -----MKLFVIL----LSLSLSLSA------------SCSTANWLIKWDDKFQNDTLSISEFKCSAALAKLGPDPKHPPTKLGEVLNF-----PHFVAA----PEAQTECGSCWKLRYK- 89

MpCP11 1 -------MKTTL----LSLGFAASVAFALPSSQGGSA---PEQYRVTHNPVYGNSSQRISDLACGGGEN--GIDTKWGYQTLGQIPNF-----DDVGAA-HVSSYNSPECGGCWYLEYK- 97

MpCP2 1 -----MKFTTTI--IALALAASTGA------------------VQLRFDNTYDNASGSMNTVACSTGAN--GLS-Q-RFPTFGSVPTF-----PHIGASSDIGGFNSPACGNCYTISFTF 86

MpCP3 1 -----MKFIAAV----ALLATSAVA------------------VQLQYDPVYDNADQSFGTVACSDGPN--GMLTK-GYSTFGSVPSY-------VGAVDTITGWNSESCGTCYQITWS- 82

MpCP6 1 MKAPALLTSLIL----TLLPLHASA------------------VTLAYDTVYDDRSRSLATVACSDGRN--GLLTR-NFTTFGSLPSF-----PRIGGAQAITGWNSSACGTCWEVTYTN 90

MpCP7 1 -----MKFTAAV----ALLATSAAA------------------VRLQYDPTYDNRDGSLGTTACSDGPN--GLITK-GFSTFGSLPNF-----PRIGAVDAITGWNSPQCGSCWQVTWN- 84

MpCP8 1 -----MKFIVAL----ALLTTSAVA------------------VQLQYDTAYDHADQSLSSVACSNGEN--GLLTK-GYTTFGSVKGTTA--STYVGAAEAITGWNSAACGNCYQIKWS- 87

MpCP10 1 -----MKFVVTI----AILATSAAA---LSVKR---RNTDGQSYKLRYVSEFDNGDYSFNNVACSNGEH--GMPAK-KLNKFGDLPRNANGVNVYGGGGFAVSGYGDEECGSCWEVYYND 102

MpCP12 1 -----MKFIAAI----AILATSAAA---LSIKR---QSADG-TLKLRYINEYDNSDFTLSDHGSC--SN--ILSPK-GINTVGDVNKSVDGVNVYPGGAFPVKEWNSDSCGTCWKVSYDD 99

CpCP 82 ----GNSIFIRGVDSGRG--GFNVNPTAFTKLVGSTE-----AGRVDNV-NYVQVDLSNCINGAN------------------------------------------------------- 134

Sm1 88 ----GHTIYVLAVDHAAS--GFNIALDAMNALTGGQA----VQLGRVSA-TATQVPVKNCGL---------------------------------------------------------- 138

MpCP1 89 AHTTPKSIYMVAIDRSAE--GFTASKQAMDDLTNKRA----EELGTVNV-DVRKVDFSRCERKS-------------------------------------------------------- 145

MpCP9 89 STGT-HTINFTAIDAGGS--DFVTGRVALDLLTNGQA----EQLGVIPV-NATAVNASACGL---------------------------------------------------------- 142

MpCP4 102 ----GNWAYIIAVDNASDEDLFVISDEANKALTRVNGRNEGIEQDIVDLDSAHEVDRACCGFNTGTQCPTALNND-----------------------G--------------------- 173

MpCP5 90 ----GNHAFVTVVDRVEEANLFVGGTDLVKNLTTFNGAPEGYDWGTAQLFSAYQVDGSCCQQNTGKQCGDP------------------------------------------------- 156

MpCP11 98 ----GNSLYVTAVDNAAGENLFVISDGAIRSLTTVNGVNEGIEKGTVDLDSAYEVDHSYCHLPDPTGNTRTGTDK----KSPSTGKKSPT---SSNNYSV-------------------- 186

MpCP2 87 -QGVTRSINLVAIDHAGN--GFNVAQAAMDELTNGNA----VALGTIDV-QSQQVARSVCGL---------------------------------------------------------- 140

MpCP3 83 --GTGKTIHVVGVDVAGN--GFNVGQRAMDDLTNGQA----VALGNIDV-TATLVDKSACGL---------------------------------------------------------- 135

MpCP6 91 SSGIAKSLNITAIDVAGA--GFNLAQVAMDELTNGHA----VEFGRVEV-TSRQVNASACGL---------------------------------------------------------- 145

MpCP7 85 --GN--SINILGMDVAGN--GFNIAQAAMDELTNGQA----VALGNIDV-QATQVDASACGL---------------------------------------------------------- 135

MpCP8 88 --GSDRTINVIAID---------------------------------------------------------------------------------------------------------- 99

MpCP10 103 -----RKVRVIVVDTAND--GFNLPKKGMDELTNGQA----YDLGVIDV-TTQKLKPADCGLNSTEGQTSMNGQTSTQGKPSTQGKTSTQEKTSTNGKALTEGKTSTNEQPPTQENTSTN 210

MpCP12 100 -NGTVRTIRVVAVDTAQE--GFNVAQKAMDELTDGKA----YEKGFVTV-TAEQLSADDCKIGTN------------------------------------------------------- 156

CpCP -----------------------------------------------------------------------------------------------------------------

Sm1 -----------------------------------------------------------------------------------------------------------------

MpCP1 -----------------------------------------------------------------------------------------------------------------

MpCP9 -----------------------------------------------------------------------------------------------------------------

MpCP4 -----------------------------------------------------------------------------------------------------------------

MpCP5 -----------------------------------------------------------------------------------------------------------------

MpCP11 -----------------------------------------------------------------------------------------------------------------

MpCP2 -----------------------------------------------------------------------------------------------------------------

MpCP3 -----------------------------------------------------------------------------------------------------------------

MpCP6 -----------------------------------------------------------------------------------------------------------------

MpCP7 -----------------------------------------------------------------------------------------------------------------

MpCP8 -----------------------------------------------------------------------------------------------------------------

MpCP10 211 KQTSTEGKTSTNGKTLTEEKTSTNEQTSTNGKALTEGKTSTNEQPPTQENASTNEQTSTEGKTLTQEKTSTNEQTPTQENTSTNGKTLTEGKAATEEQTATEGKTSTEGMTAS 323

MpCP12 -----------------------------------------------------------------------------------------------------------------

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Supplemental Table 2

Data Collection

MpCP1 (3SUJ) MpCP2 (3SUK) MpCP3 (3SUL) MpCP5 (3SUM)

Beamline, at LNLS - Brazil W01B-MX2 W01B-MX2 D03B-MX1 D03B-MX1

Wavelength (Å) 1.4586 1.4586 1.4299 1.4370

Space group P43212 P1211 P3121 P212121

Cell parameters a, b, c (Å) 45.3, 45.3, 105.74

29.5, 86.0, 43.6, (β=106.8o)

48.3, 48.3, 94.0 49.7, 107.9, 112.7

Resolution range (Å) 14.61-1.34 (1.41-1.34)

14.62-1.34 (1.41-1.34)

14.11-1.63 (1.72-1.63)

23.02-1.87 (1.97-1.87)

Unique reflections 25598 (3654) 43181 (5984) 16287 (2314) 50913 (7306)

Average redundancy 39.3 (36.9)a 8.3 (8.2) 13.2 (13.2) 6.3 (6.0)

Rsymm (%) 8.5 (51.8) 6.5 (27.0) 5.4 (59.0) 8.4 (32.7)

Completeness (%) 99.8 (100)a 92.4 (87.7) 99.2 (98.4) 99.8 (99.6)

I/σ(I) 42.1 (11.7) 22.2 (8.9) 25.5 (4.3) 13.2 (4.4)

Monomers per asymmetric unit 1 2 1 4

Solvent content (%) 37.3 40.33 50.88 51.28

Number of sites found (expected) 8 (7) - - -

Mean map CC (%) 76.7 - - -

Refinement

Resolution range (Å) 14.51-1.34 14.50-1.34 13.98-1.63 20.00-1.87

Reflections (cross-validationb) 24215 (1299) 41002 (2177) 15419 (825) 48195 (2571)

Rfactor/Rfree (%) 18.43/22.00 17.32/20.50 23.77/26.77 16.51/22.33 Average B-factor/r.m.s.d. (Å2)

main chain (no. of residues) side chain (no. of residues) ligands (no. of molecules) solvent (no. of molecules)

11.56/0.86 14.93/2.18

19.96 26.63

10.86/0.79a 12.87/1.59a

- 24.6

20.76/0.99 22.56/1.96

- 30.73

19.25/0.79b 21.46/2.21b

- 31.13

R.m.s.d. from standard geometry bond length (Å) bond angles (°)

0.025 2.254

0.024 1.698

0.023 2.23

0.022 1.887

Ramachandran plot most favored (%) allowed (%) outlier (%)

95.7 3.5 0.8

96.6 2.5 0.9

94.1 4.2 1.7

96.8 2.5 0,7

a Friedel pairs treated separately. b Cross-validation test set size of 5%. Data for outer shell shown in parentheses.

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Supplemental Table 3 MpCP1 fragments detected in the flow through fraction (in red):

a Score > 5 was considered significative by Mascot (Perkins et al. 1999) analysis.

Peptide

Number

(MpC1)

Peptide sequence identified m/z Charge

Number of

times

identified

Scorea

1 SAVKLSYD 882,4640 +1 1 14

2 FSRCERKS 535,2494 +2 3 12

3 SAVKLSYDEAY 632,3006 +2 2 32

4 SAVKLSYDEAYD 680,7709 +2 10 45

5 NGLYPKYRTFGD 715,8497 +2 3 40

6 GENGLYPKYRTFGD 808,8441 +2 5 48

7 VRKVDFSRCERKS 833,9015 +2 2 9

8 VRKVDFSRCERKS 556,2705 +3 1 6

9 NVDVRKVDFSRCERKS 665,6593 +3 1 12

10 SAVKLSYDEAYDNPSSSLL 1029,9834 +2 3 53

11 SAVKLSYDEAYDNPSSSLLS 1073,4673 +2 1 21

12 TVNVDVRKVDFSRCERKS 732,3623 +3 1 4

13 LTNKRAEELGTVNVDVRKVD 752,7372 +3 1 27

14 GENGLYPKYRTFGDLPGFPCIGGSSD 935,4387 +3 1 14

15 NPSSSLLSVTCSDGENGLYPKYRTFGD 988,7849 +3 2 54

16 RSAEGFTASKQAMDDLTNKRAEELGTVNVD 1085,1835 +3 1 2

17 EAYDNPSSSLLSVTCSDGENGLYPKYRTFGD 1148,1814 +3 1 13

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MpCP2 fragments detected in the flow through fraction (in red):

Peptide

Number

(MpCP2)


Number of

times

identified

Scorea

1 SDIGGFN 709,3103 +1 4 21

2 SINLVAI 729,4543 +1 1 16

3 PTFPHIG 384,7053 +2 2 24

4 SGAVQLRFD 496,7491 +2 2 61

5 PTFPHIGASSD 564,7704 +2 2 47

6 TIDVQSQQVAR 622,8301 +2 2 75

7 TFGSVPTFPHIG 630,3252 +2 3 13

8 GTIDVQSQQVAR 651,3428 +2 2 92

9 SGAVQLRFDNTY 685,8399 +2 4 46

10 LGTIDVQSQQVAR 707,8842 +2 1 84

11 SGAVQLRFDNTYD 743,3260 +2 14 64

12 SINLVAIDHAGNGFN 771,3869 +2 2 42

13 SGAVQLRFDNTYDNA 835,8828 +2 3 62

14 SINLVAIDHAGNGFNVA 856,4368 +2 4 11

15 NVAQAAMDELTNGNAVAL 901,4390 +2 2 9

16 SGAVQLRFDNTYDNASG 907,9082 +2 3 80

17 GNAVALGTIDVQSQQVAR 913,9869 +2 3 73

18 SINLVAIDHAGNGFNVAQ 920,4665 +2 3 48

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19 SINLVAIDHAGNGFNVAQA 955,9877 +2 1 9

20 NGNAVALGTIDVQSQQVAR 971,0080 +2 1 84

21 ANGLSQRFPTFGSVPTFPHIGASSD 864,0925 +3 5 15

22 AAMDELTNGNAVALGTIDVQSQQVAR 891,4412 +3 4 58

23 STGANGLSQRFPTFGSVPTFPHIGASSD 945,7907 +3 3 10

24 VAQAAMDELTNGNAVALGTIDVQSQQVAR 990,8266 +3 2 23

25 NVAQAAMDELTNGNAVALGTIDVQSQQVAR 1028,8452 +3 4 10



Peptide

Number

(MpCP3)


Number of

times

identified

Scorea

1 VTATLVD 718,3926 +1 2 13

2 GPNGMLTK 409,2140 +2 2 34

3 GPNGMLTKG 437,7297 +2 2 22

4 SVAVQLQY 907,4876 +1 2 8

5 GPNGMLTKGY 519,2561 +2 2 48

6 VAGNGFNVGQR 559,7901 +2 3 31

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7 GPNGMLTKGYS 562,7736 +2 1 38

8 TGKTIHVVGVD 563,3158 +2 2 30

9 PVYDNADQSFG 1212,5112 +1 1 47

10 PVYDNADQSFG 606,7616 +2 1 29

11 GPNGMLTKGYSTF 686,8341 +2 2 56

12 GPNGMLTKGYSTFG 715,3410 +2 4 78

13 VAGNGFNVGQRAMD 718,3234 +2 3 73

14 VAGNGFNVGQRAMDD 775,8395 +2 4 76

15 SVAVQLQYDPVYDNAD 898,9180 +2 19 18

16 GPNGMLTKGYSTFGSVPS 900,4334 +2 1 9

17 GPNGMLTKGYSTFGSVPSYVG 1060,0060 +2 4 33

18 GPNGMLTKGYSTFGSVPSYVGAV 1145,0629 +2 3 7

19 PNGMLTKGYSTFGSVPSYVGAVD 1174,0703 +2 1 10

20 GPNGMLTKGYSTFGSVPSYVGAVD 1202,5554 +2 2 71

21 GPNGMLTKGYSTFGSVPSYVGAVDTITG 1388,6716 +2 3 15

22 VAGNGFNVGQRAMDDLTNGQAVALGNID 939,7775 +3 5 30

23 VAGNGFNVGQRAMDDLTNGQAVALGNID 1409,1759 +2 2 22

24 GPNGMLTKGYSTFGSVPSYVGAVDTITGWNSE 1098,1767 +3 4 32



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Peptide

Number

(MpCP5)

Peptide sequence identified m/z Charge Number of

times identified Score

a

1 SNWLIK 760,4230 +1 1 37

2 WLIKWD 860,4585 +1 1 22

3 PKHPPTKL 459,2786 +2 1 11

4 SAALAKLGPD 942,5171 +1 2 45

5 SNWLIKW 473,7583 +2 2 17

6 SNWLIKW 946,5098 +1 3 16

7 PKHPPTKLG 487,7912 +2 1 13

8 SNWLIKWD 531,2532 +2 4 30

9 SNWLIKWD 1061,5071 +1 2 41

10 PKHPPTKLGE 552,3131 +2 1 11

11 SNWLIKWDD 588,7675 +2 3 35

12 SNWLIKWDD 1176,5611 +1 2 39

13 SNWLIKWDDK 652,8315 +2 2 22

14 PKHPPTKLGEVL 658,3866 +2 2 30

15 LVKNLTTFNGAPEG 730,8922 +2 1 6

16 PKHPPTKLGEVLNF 526,2931 +3 1 2

17 WLIKWDDKFQND 804,3898 +2 3 11

18 LVKNLTTFNGAPEGY 812,4177 +2 3 6

19 LVKNLTTFNGAPEGYD 869,9199 +2 3 20

20 SNWLIKWDDKFQND 603,6128 +3 4 32

21 SNWLIKWDDKFQND 904,9174 +2 6 35

22 PKHPPTKLGEVLNFPH 604,3268 +3 4 25

23 PKHPPTKLGEVLNFPHF 653,3468 +3 3 49

24 WDDKFQNDTLSISEFK 658,3108 +3 1 21

25 TLSISEFKCSAALAKLGPD 670,0140 +3 1 39

26 PKHPPTKLGEVLNFPHFVAAPE 809,0981 +3 1 5

27 PKHPPTKLGEVLNFPHFVAAPEAQ 875,4647 +3 3 17

28 KFQNDTLSISEFKCSAALAKLGPD 880,7803 +3 2 34

29 GNHAFVTVVDRVEEANLFVGGTDLVK 929,4800 +3 5 28


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Supplemental Figure S1

.

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Supplemental Figure S3A

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Supplemental Figure S3B

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Supplemental Figure S3C

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Supplemental Figure S3D

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Supplemental Figure S3E

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Documents

Functional diversification of cerato-platanins in Moniliophthora … · 2019. 12. 20. · Other cerato-platanins present diverse biological activities, such as the induction of systemic