Disruption and molecular characterization of calpains-related (MoCAPN1, MoCAPN3 and MoCAPN4) genes in Magnaporthe oryzae

M

D(

IFa

b

c

d

a

ARRAA

KMCPGC

1

bfl1ftdpgaer2

h0

ARTICLE IN PRESSG ModelICRES-25668; No. of Pages 11

Microbiological Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Microbiological Research

j ourna l h om epage: www.elsev ier .com/ locate /micres

isruption and molecular characterization of calpains-relatedMoCAPN1, MoCAPN3 and MoCAPN4) genes in Magnaporthe oryzae

rshad Ali Khana,c, Yao Wanga, Hai-Jiao Lia, Jian-Ping Lub, Xiao-Hong Liua,u-Cheng Lina,d,∗

State Key Laboratory for Rice Biology, Biotechnology Institute, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, ChinaSchool of Life Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, ChinaDepartment of Plant Pathology, Faculty of Crop Protection Sciences, The University of Agriculture, Peshawar, PakistanChina Tobacco Gene Research Center, Zhengzhou Tobacco Institute of CNTC, Zhengzhou 450001, China

r t i c l e i n f o

rticle history:eceived 13 May 2013eceived in revised form 13 March 2014ccepted 20 March 2014vailable online xxx

eywords:agnaporthe oryzae

onidiationathogenicityrowth

a b s t r a c t

Calpains are intracellular, cysteine proteases found in plants, animals and fungi functioning as signaltransduction components in different cellular pathways including sporulation and alkaline adaptationin fungi. Calpains-related MoCAPN1 (MGG 14872), MoCAPN3 (MGG 15810) and MoCAPN4 (MGG 04818)genes from Magnaporthe oryzae genome which are 2604, 3513 and 771-bp in length and encoding iden-tical proteins of 867, 1170 and 256 amino acids were functionally characterized for different phenotypesthrough gene disruption method. All the mutants except those for MoCAPN1 showed normal phenotypes.In pathogenicity test, the mutants did not lead to any visible changes in phenotypes causing similar blastlesions on blast susceptible rice and barley leaves as those of the Guy-11 strain suggesting no major rolein pathogenicity. Germ tubes formation, appressorium formation, mycelium radial growth and matingwith 2539 strain were indistinguishable among the mutants and Guy-11 strains. Cell wall integrity (congo

alpains red) test, stress response under chemical pressure (ZnSO4, CuSO4 and CdCl2), osmotic and oxidative (NaCland H2O2) stress response, growth response on glucose and nitrogen deficient media resulted in similarresults in the mutants and Guy-11 strains. However, mutants for �MoCAPN1 gene produced reduced(0.57 ± 0.15B and 0.54 ± 0.05B) conidia compared to that (1.69 ± 0.13A) of the Guy-11 strain showing itsinvolvement in conidiation.

. Introduction

Magnaporthe oryzae, the causal agent of the devastating ricelast disease, is a grave threat to global rice production, reportedrom about 85 countries worldwide causing 70–80% of rice yieldosses during an epidemic season (Ou, 1985; Howard and Valent,996; Baker et al., 1997). M. oryzae, an ascomycete phytopathogenicungus with both scientific and economical value, is also an impor-ant model fungus for molecular-based studies of fungal-causediseases. Rice is one of the most important food security cropsroviding 23% calories to mankind. Both M. oryzae and rice areenetically tractable and their genome sequence information isvailable. It has been reported that each year M. oryzae destroys

Please cite this article in press as: Khan IA, et al. Disruption and moland MoCAPN4) genes in Magnaporthe oryzae. Microbiol Res (2014), htt

nough rice to feed 60 million people (Zeigler et al., 1994). Recently,ice blast has caused epidemics in China during years 2001 and005, where 5.7 million hectares of rice were destroyed. During

∗ Corresponding author. Tel.: +86 571 88982291; fax: +86 571 88982183.E-mail address: [email protected] (F.-C. Lin).

ttp://dx.doi.org/10.1016/j.micres.2014.03.003944-5013/© 2014 Elsevier GmbH. All rights reserved.

© 2014 Elsevier GmbH. All rights reserved.

1975 and 1990, the rice blast disease caused a loss of 157 milliontons of rice all over the world (Veneault-Fourrey and Talbot, 2005).

M. oryzae causes infection through a specialized dome-shapedappressorium penetrating the host tissue through a penetrationpeg by using turgor pressure derived from lipid and glyceroldegradation. Studies have shown that appressorium-mediatedpenetration events are controlled by highly conserved sev-eral signaling networks (G protein coupled receptors (GPCRs),autophagy, mitogen-activated protein kinase (MAPK), cyclicadenosyl monophosphate (cAMP) and Ca2+-mediated signaling)and these signalings are regulated by genes (Adachi and Hamer,1998; Lee and Dean, 1993; Kang et al., 1999; Xu and Hamer, 1996;Choi and Dean, 1997; DeZwaan et al., 1999; Clergeot et al., 2001;Kim et al., 2005; Zhao et al., 2005; Veneault-Fourrey et al., 2006;Odenbach et al., 2007; Mitchell and Dean, 1995; Liu and Dean, 1997;Thines et al., 2000; Park et al., 2006; Zhao and Xu, 2007; Liu et al.,

ecular characterization of calpains-related (MoCAPN1, MoCAPN3p://dx.doi.org/10.1016/j.micres.2014.03.003

2009). The disruption of such signaling genes results in the lossand/or reduction of pathogenicity as well as pleiotrophic effects oncellular processes including mating, conidiation and growth rate.The components of these signalings may represent targets for the

dx.doi.org/10.1016/j.micres.2014.03.003


http://www.sciencedirect.com/science/journal/09445013

http://www.elsevier.com/locate/micres

mailto:[email protected]


ING ModelM

2 ical R

dinr

iavtotp

(pdoHCiaopistemcAi1

ih(oiFtettbfi

fiui

lgmi(s(o

Mnatri

ARTICLEICRES-25668; No. of Pages 11

I.A. Khan et al. / Microbiolog

evelopment of antifungal drugs. Although several genes regulat-ng these signalings have been characterized, further research iseeded to get a better understanding of the biology and signallingegulated genes to effectively control this fatal disease.

Pathogenicity-related genes are of great interest not only toncrease our understanding of infection process, but also becauseny such gene could become a target for disease control. Keeping iniew the worldwide importance of this blast disease and to knowhe function of calpains-related genes in the blast disease of M.ryzae, we characterized three calpains-related genes and namedhem MoCAPN1, MoCAPN3 and MoCAPN4 based on their homologyrotien sequence in human beings.

Calpains, a family of well conserved calpain-related proteinsC+2-regulated cysteine proteases) playing an important role inhysiological and pathological processes (a critical feature of cellivision cycle) have been reported in all prokaryotic and eukary-tic organisms including fungi, plants and protists (Franco anduttenlocher, 2005; Goll et al., 2003; Lebart and Benyamin, 2006;roall and Ersfeld, 2007). Intracellular proteases play a critical role

n the regulation of metabolism. Calpains were originally identifieds calcium-dependent proteolytic enzymes in a study conductedn rat and were later named calpains derived from calcium andapain (Guroff, 1964). A distinguishing feature of calpains activity

s their ability to confer limited cleavage protein substrates intotable fragments rather than complete proteolytic digestion andhis calpain-mediated proteolysis is a major pathway that influ-nces various aspects of cell physiology, including apoptosis, celligration and cell proliferation (Sorimachi et al., 1997). Role of

alpains has also been implicated in human pathological disorders. study has shown that an elevated calpain activity was detected

n breast cancer tissues compared to normal ones (Shiba et al.,996).

The role of calpains in invasive process has also been reportedn cancer prostate cells (Mamoune et al., 2003). Although calpainsave been reported to play important roles in human pathogenesisHuang and Wang, 2001), however, no studies have been reportedn calpains role in fungal pathogenesis and further investigations needed to find their role in fungal developmental processes.urthermore, the role of calpains in sporulation and alkaline adap-ation has also been reported in Saccharomyces cerevisiae (Futait al., 1999). Conidiation is an important developmental processhat plays a central role in the life cycles of M. oryzae. The role of cys-ein proteases has also been reported in the formation of spores inacteria (Kornberg et al., 1968), ascospores inyeasts (Geisen, 1993),ruiting bodies in slime molds (North, 1982) and conidial dischargen fungi (Phadatare et al., 1997).

To find the role of these genes in blast disease, we firstly identi-ed these genes using bioinformatics analysis and then disruptedsing gene disruption method. The mutants after mono-conidial

solation were finally confirmed by southern blotting.The mutants for all the deleted genes were functionally ana-

yzed for the rice blast-related phenotypes such as mycelium radialrowth on CM medium (9 dpi), conidiation (9 dpi), conidial ger-ination (2 and 4 hpi), appressorium formation (6 hpi), cell wall

ntegrity (congo red) test, stress response under chemical pressureZnSO4, CuSO4 and CdCl2), osmotic and oxidative (NaCl and H2O2)tress response, pathogenicity test (rice and barley) and mating test2539 strain) and all these phenotypes were compared with thosef the wild-type pathogenic Guy-11 strain.

Mutants deleted for the genes MoCAPN1, MoCAPN3 andoCAPN4 showed normal phenotypes such as full pathogenicity,

ormal culture colony growth, un-altered conidial germination,


ppressorium formation and mating with 2539 strain similar tohose of the Guy-11 indicating that these genes are not involved inice blast disease. However, reduction in conidiation was observedn the mutants of MoCAPN1 gene.

PRESSesearch xxx (2014) xxx–xxx

We conclude that MoCAPN1, MoCAPN3 and MoCAPN4 are notessential for the blast fungus to cause pathogenicity. Furtherresearches at molecular basis that are necessary for understand-ing the molecular machinery of this fungal pathogen will result inmore durable control strategies of this disease.

2. Materials and methods

2.1. Growth conditions of fungal strains

Rice-infecting M. oryzae wild-type Guy-11 and mutants for allthe deleted genes were cultured on complete medium (CM, 10 gglucose, 2 g peptone, 1 g yeast extract, 1 g casamino acids, 0.1%(v/v) trace element, 0.1% (v/v) vitamin supplement, 6 g NaNO3,0.52 g KCl, 0.52 g MgSO4, 1.52 g KH2PO4 and 1 L water, pH 6.5)at 25–28 ◦C for 9–12 days with a 14 h light and 10 h dark cycleusing fluorescent lights (Liu et al., 2007). For growth analysis, thestrains were grown on minimal medium (MM, 6 g NaNO3, 0.52 gKCl, 0.52 g MgSO4, 1.52 g KH2PO4, 10 g glucose, 0.5% biotin and 1 Lwater, pH 6.5), MM-N (MM medium without the nitrogen source),and MM-C medium (MM medium without the carbon source) at25–28 ◦C for 9–12 days under light conditions. The defined com-plex medium (DCM, yeast nitrogen base without amino acid 1.7 g,NH4NO3 2 g, l-asparagine 1 g and glucose 10 g, Na2HPO4 was usedto adjust pH to 6.0, and 1 L water) supplemented with 100 �g/mLchlorimuron as antibiotic (Roche, Germany) was used for initialscreening of chlorimuron-resistant mutants during ATMT transfor-mation as described previously (Liu et al., 2008). All the Guy-11and mutants were stored by growing the fungus on sterile filterpaper discs over the CM medium at 25–28 ◦C for 9–12 days fol-lowed by desiccation in vacuum for 7 days and storage (−20 ◦C) forfuture use. Escherichia coli strain DH5� and Agrobacterium tumefa-ciens strain AGL-1 were cultured in an LB containing ampicilin andkanaymcyin at 37 and 28 ◦C, respectively, for transformation. Mat-ing type 2539 strain (Mat1-1) was used for sexual crosses on OMA(Oat Meal Agar) medium plate. Guy-11 and the mutants genomicDNA was extracted from fungal mycelia using 2% CTAB methodby growing the strains in liquid CM medium at 28 ◦C for 4–5 days(150–200 rpm) as described previously (Talbot et al., 1993). Poly-merase chain reactions (PCR), restriction enzyme digestions, gelelectrophoresis and ligation reactions were carried out followingstandard procedures (Sambrook and Russell, 2002).

2.2. PCR cloning and bioinformatics analysis of MoCAPN1,MoCAPN3 and MoCAPN4

M. oryzae genome database (http://www.broad.mit.edu)was used to locate the gene locii corresponding to MoCAPN1(MGG 14872), MoCAPN3 (MGG 15810) and MoCAPN4(MGG 04818). The null mutants were then constructed bygene disruption method as previously described (Sambrook andRussell, 2002). Primers used in this study were designed by usingwww.idtdna.com and commercially synthesized (Sangon Co.,Shanghai, China).

2.3. Targeted gene disruption and spore PCR assay

For constructing the MoCAPN1, MoCAPN3 and MoCAPN4 genesreplacement vectors, an up and down flanking sequences of thesethree genes were amplified by PCR from the genomic DNA of Guy-11 and inserted into the vector pBS-SUR/NEO/HPH respectively(Li et al., 2011). Up fragments (1.1, 1.0 and 1.2-kb) in MoCAPN1,


MoCAPN3 and MoCAPN4 genes were PCR amplified with primersCAPN1-up-p1: 5′-CCGGGAGGGGGTAAGTCG-3′ and CAPN1-up-p2: 5′-AAgatatcGGGCGCCAGAATCACAGTC-3′, CAPN3-up-p1:5′-GTctcgagGAAGACGCGAGGAGGAAGGTTA-3′ and CAPN3-up-p2:


http://www.broad.mit.edu/

http://www.idtdna.com/

ING ModelM

ical R

555XpDgA5555pbStlapbtAr(spa3CCCibb

2

dp(dewatpnaIa(m8uwmfhft

2

i



′-GTgatatcCGCGTACTGGCACTGGTTGT-3′ and CAPN4-up-p1:′-TTctcgagAAATCCAAAACTCCCCATCATCAT-3′ and CAPN4-up-p2:′-TTgtcgacAAGAAGGGCGTCGTAATCAAC-3′ and cloned betweenhoI and EcoRV, XhoI and EcoRV and Xho1 and Sal1 sites of vectorBS-SUR/NEO/HPH to generate pBS-CAPN1/CAPN3/CAPN4-up.own fragments (1.5, 1.5 and 1.2-kb) of CAPN1, CAPN3 and CAPN4enes, after PCR amplified with primers CAPN1-low-p1: 5′-AggatccGTGCGCCCGAGGTAGCTTTTCTTG-3′ and CAPN1-low-p2:′-AAtctagaCACGCGCCTTATCTATGGGGACAG-3′, CAPN3-low-p1:′-AAggatccTCACCGCGGCTGGAGACAATACCC-3′, CAPN3-low-p2:′-AAtctagaCCAACGCCCCGCAGACACCTTC-3′, and CAPN4-low-p1:′-GtaagcttAAACGCCAAGCCAAGCCAATCAGG-3′ and CAPN4-low-2: 5′-GtcccgggCGAAACACTACCGCCGGGAGAACA-3′ were insertedetween the BamH1 and Xba1, BamH1 and Xba1, and HindIII andma1 sites of vector pBS-CAPN1/CAPN3/CAPN4-down to generatehe final replacement vector pBS-up-CAPN1/CAPN3/CAPN4-ow. The pBS-up-CAPN-low was then Xho1/Xba1, Xho1/Xba1nd Xho1/Sma1 linearized and 5.4, 3.5 and 3.8-kb fragmentsurified by gel electrophoresis were transformed to a donorinary vector pCAMBIA-1300. The vector pCAMBIA-1300 washen transformed to the conidia of the Guy-11 strain using an. tumefaciens AGL-1 strain for transformation. Knock-out chlo-imuron (100 �g/mL), geneticin (800 �g/mL) and hygromycin400 �g/mL) resistant transformants were initially screened onelective media (DCM and CM) and then confirmed by PCR usingrimers CAPN1-check-p1: 5′-ATGCATACTCCATCCTCCAAGCCA-3′

nd CAPN1-check-p2: 5′-ACCTCGAGCAAGAACTTGGACTGT-′, CAPN3-check-p1: 5′-TGCTTTCGACCCGCTGAGATGTAT-3′,APN3-check-p2: 5′-AATGCCTGTGCTGCATCCAAAGAG-3′ andAPN4-check-p1: 5′-GCCAACATGTGGCCGTTCAAGTTA-3′ andAPN4-check-p2: 5′-ACAAGGTCGCATTCTCTCAGCACT-3′ resulting

nto 415, 342 and 551-bp bands. Knock-out mutants were purifiedy mono-conidial isolation and after confirmed by southernlotting were used for phenotypic analysis.

.4. Transformation of M. oryzae

A. tumefaciens-mediated fungal transformation was con-ucted by growing the A. tumefaciens strain AGL1 containingCAMBIA-1300 vector in liquid LB medium containing kanamycin100 �g/mL) at 28 ◦C for 12 h in shaker incubator as previouslyescribed (Sweigard et al., 1992; Wu et al., 2006). A 300 �L A. tum-ficiens overnight-grown cells (optical density OD600 at 600 nm)ere incubated in 5 mL induction medium (IM) containing 200 �M

cetosyringone (AS) at 28 ◦C for an additional 6 h. A 100 �L A.umefaciens culture after mixed with equal volume of conidial sus-ension (106 conidia/mL) of M. oryzae was pipette-dropped over aylon membrane (3 cm × 3 cm) (Millipore Co., Bedford, MA, USA)nd equally spread with sterilized scalpel followed by plating onM medium containing 200 �M AS. The membrane after incubatedt 22 ◦C for 2 days under dark conditions was cut into small pieces3 cm × 0.1 cm) and placed upside down over solid DCM and CM

edium containing chlorimuron, geneticin and hygromycin (100,00 and 400 �g/mL) as antibiotics. After grown for 4–7 days at 28 ◦Cnder dark conditions, the antibiotic-resistant colonies appearedere transferred to a fresh DCM/CM medium containing the aboveentioned antibiotics for growth followed by incubation at 28 ◦C

or 4–7 days under light conditions. The chlorimuron, geneticin andygromycin-resistant transformants after PCR confirmation were

urther purified by mono-conidial isolation for subsequent pheno-ypic analysis.


.5. Nucleic acid isolation and southern blotting

Genomic DNA from all the knock-out and Guy-11 strains wassolated from mycelia grown in liquid CM medium using 2% CTAB

PRESSesearch xxx (2014) xxx–xxx 3

as previously described (Talbot et al., 1993). To perform southernblotting, genomic DNA of the mutants and Guy-11 were digestedwith Sal1, EcoRV and Stu1, separated on 0.7% agarose gel andtransferred to a positively charged nylon membrane. Using the dn-fragment (1.5-kb), up-fragment (1.0-kb) and dn-fragment (1.2-kb)of MoCAPN1, MoCAPN3 and MoCAPN4 genes respectively as probes,obtained by PCR from genomic DNA of Guy-11 and Sal1, EcoRV andStu1 as enzymes, the deleted mutants showed 7.3, 4.0 and 2.8-kbbands compared to 2.4, 7.0 and 1.7-kb bands in the Guy-11 strainin MoCAPN1, MoCAPN3 and MoCAPN4 genes respectively.

2.6. Assays for mycelium radial growth and conidiation

To analyze the vegetative mycelium radial colony growth char-acteristics, a 5 mm mycelium block of 8–12-day-old Guy-11 andmutants was inoculated on the center of solid CM medium followedby incubation at 25–28 ◦C for 9 days under constant fluorescentlight. Diameters (cm) of the mycelium colony were recorded andphotographed (Canon DS126231, Taiwan) using the proceduredescribed previously (Talbot et al., 1993).

Conidiation was carried out by harvesting the whole coni-dia of a 9-day-old CM-grown culture of the Guy-11 and mutantswith 5 mL sterile distilled water by gently rub the surface ofthe plate with a sterile Q-tip. A 10 �L conidial suspension wasdropped on a clean microscope glass coverslip and spore con-centration was determined by using the formula (total average ofconidia × 50/3.14 × (total average of colony diameter)2 × 25) usinghemacytometer under a microscope (Nikon YS 100 Japan). Thisassay was repeated three times in triplicate independently.

2.7. Analysis for conidial germination and appressoriumformation

To measure conidial germination and appressorium formation, a20 �L drop of conidial suspension (105 conidia mL−1) was droppedon the surface of a sterile plastic coverslip and incubated in amoistened box at 25 ◦C for 2, 4 and 6 h using method as previ-ously described (Liu et al., 2007). The coverslip was then gentlyinverted and viewed under a Nikon YS100 microscope (Nikon,Japan). The percentage of the conidia that produced germ tubesand the percentage of the germ tubes that formed appressoriawere calculated using a hemacytometer. Each assay was inde-pendently repeated three times with three replicates each timewith a counting of the total number 200 conidia for each repli-cation.

2.8. Assays for mycelium radial growth on MM, MM-N and MM-Cmedium

To find the effect of nitrogen and glucose deficiency on thegrowth of deleted mutants, a 5 mm mycelium block of 8–12-day-old Guy-11 and mutants was inoculated on MM, MM-C(MM medium without glucose) and MM-N (MM medium with-out nitrogen) medium plate followed by incubation at 25–28 ◦Cfor 8 days under constant fluorescent light. Diameters (cm) ofthe mycelium colony were recorded and photographed (CanonDS126231, Taiwan) using the procedure described previously (Liuet al., 2011). The tests were repeated at least three times for eachcondition.

2.9. Cell wall integrity test (congo red)


Cell wall composition in M. oryzae plays an important role in fullvirulence and its defect can affect appressorium formation result-ing in defective pathogenicity (Xu et al., 1998; Xu, 2000; Dou et al.,2011). Cell wall integrity test was conducted by growing a 5 mm


ARTICLE IN PRESSG ModelMICRES-25668; No. of Pages 11

4 I.A. Khan et al. / Microbiological Research xxx (2014) xxx–xxx

F owed

mctlpTe

FXmvuoc

ig. 1. CysPc domain was detected in MoCAPN1 and MoCAPN3. EFh domain was sh

ycelium plug of each Guy-11 and mutants on CM medium plateontaining Congo red (Sangon Co., Shanghai, China) at concentra-ion 200 �g/mL followed by incubation at 28 ◦C for 8 days underight conditions. Diameters of fungal colonies were measured and


hotographed 8 days post-inoculation (Canon DS126231, Taiwan).his experiment was conducted three times with three replicationsach time for each strain.

ig. 2. Gene disruption vector, southern blotting, Check PCR and culture colony growth for = XbaI, S = SalI, Sm = Sma1, H = HindIII, BT = Beta Tubulin. (A,B) The MoCAPN1 gene locus,

ants. DNA of Guy-11 and mutant were digested with Sal1 and probed with 1.5-kb dn-fector map and southern blot analysis of MoCAPN3 knock-out transformants. Genomicp-fragment amplified with primers CAPN3-Up-P1 and CAPN3-Up-P2. (E) The MoCAPN4f MoCAPN4. DNA of Guy-11 and mutants were digested with Stu1 and probed with 1.2-konfirmation for deleted mutants of MoCAPN4.

in MoCAPN4. Conserved domains were drawn at http://smart.embl-heidelberg.de/.

2.10. Stress response under chemicals pressure (ZnSO4, CuSO4and CdCl2)

To evaluate the vegetative growth of the wild-type strain Guy-


11 and mutants under chemical stress condition, 5 mm myceliumblock of each strain was grown on solid MM medium supplementedwith ZnSO4 (10 mM), CuSO4 (1 mM) and CdCl2 (0.5 mM) followed

�MoCAPN1, �MoCAPN3 and �MoCAPN4 mutants. Xh = XhoI, EV = EcoRV, B = BamHI,targeting disruption vector and southern blotting of MoCAPN1 knock-out transfor-ragment amplified with primers CAPN1-dn-P1 and CAPN1-dn-P2. (C,D) Disruption

DNA of Guy-11 and mutants were digested with EcoRV and probed with 1.0-kb gene locus, targeting disruption vector and PCR confirmation for deleted mutantsb dn-fragment amplified with primers CAPN4-dn-P1 and CAPN4-dn-P2. (F) RT-PCR


http://smart.embl-heidelberg.de/


I.A. Khan et al. / Microbiological Research xxx (2014) xxx–xxx 5

etative

bar(t

2

Gbpbaepui

2

aocbaw

Fig. 3. Growth phenotypes of the strains on CM medium. (A–C) Veg

y incubation at 25–28 ◦C for 8 days under dark conditions using method described previously (Tucker et al., 2004). Diameters ofepresentative fungal colonies were measured and photographedCanon DS126231, Taiwan). This experiment was repeated threeimes with three replicates each time for each strain.

.11. Osmotic and oxidative stress response (NaCl and H2O2)

To find the hyperosmotic and oxidative stress response of theuy-11 and mutants against NaCl and H2O2, a 5 mm myceliumlock of each Guy-11 and mutant strain was grown on solid com-lete medium containing NaCl (1 M) and H2O2 (5 mM) followedy incubation at 25–28 ◦C for 8 days under light conditions using

method described previously with little modification (Zhangt al., 2011). Diameters of fungal colonies were measured andhotographed (Canon DS126231, Taiwan) 8 days post-inoculationsing measuring tap. This experiment was repeated three times

ndependently with three replicates each time for each strain.

.12. Infection assays (rice and barley)

The virulence test on rice and barley seedlings was performeds described previously (Lu et al., 2007; Talbot et al., 1993). 14-day-ld rice and 7-day-old barley seedlings were inoculated with 10 mL


onidial suspension (5 × 104 conidia/mL) and 5 mm myceliumlocks of Guy-11 and mutants and incubated in growth chambert 25–28 ◦C for 7 and 4 days. Controlled plants were inoculatedith gelatin solution only. Rice and barley disease lesions were

growth of deletion mutants on CM medium at 25–28 ◦C for 9 days.

photographed 7 and 4 days post-inoculation using canon camera(Canon DS126231, Taiwan). This experiment was repeated threetimes independently.

2.13. Mating with 2539 strain

The role of deleted gene in sexual reproduction was determinedby crossing the mutant and Guy-11 with the opposite mating typestrain 2539 for perithecia formation as previously described (Liet al., 2010). Mycelium blocks (5 mm) of each Guy-11, mutants and2539 strains were placed on OMA medium plate at about 5 cm apartfrom one another and incubated at 25–28 ◦C under light conditionsfor about a week until their colonies merge and then transferred toanother incubator at 22 ◦C under constant fluorescent light for 28days. Photographs were taken after 4 weeks post-incubation witha canon camera (Canon DS126231, Taiwan). Perithecia productionat the junctions between mated individuals shows that the gene isnot essential for sexual reproduction in M. oryzae.

3. Results

3.1. Isolation of MoCAPN1, MoCAPN3 and MoCAPN4

Blastp searches (http://www.broad.mit.edu) against M. oryzae


showing orthologs (MGG 14872, MGG 15810 and MGG 04818) instrain 70-15 were designated as MoCAPN1, MoCAPN3 and MoCAPN4based on their homologous protein sequence in Homo sapiens.Multiple alignment of these genes resulted in several homologs


http://www.broad.mit.edu/



Table 1Comparision of radial growth, conidiation, germination and appressorium formation among mutants of M. oryzae.

Strains Growth (cm)a Conidiation (×103/mm2)b Germination (%)c Appressorium formation (%)d

2 hpi 4 hpi 6 hpi

Guy-11 6.57 ± 0.05A 1.69 ± 0.13A 93.66 ± 1.15A 98.83 ± 2.51A 92.66 ± 2.08ACAPN1-16 6.65 ± 0.05A 0.57 ± 0.15B 96.16 ± 2.08A 99.5 ± 1.73A 94.83 ± 4.16ACAPN1-35 6.67 ± 0.05A 0.54 ± 0.05B 95.83 ± 3.21A 99.83 ± 0.57A 95.16 ± 2.51ACAPN3-4 6.47 ± 0.05A 1.53 ± 0.22AB 93.33 ± 4.04A 99.83 ± 0.57A 93.66 ± 1.15ACAPN3-7 6.55 ± 0.05A 1.3 ± 0.14AB 93.16 ± 1.15A 99.83 ± 0.57A 93.5 ± 2ACAPN4-9 6.55 ± 0.05A 1.08 ± 0.13AB 94.83 ± 8.73A 99.33 ± 1.15A 92.16 ± 0.57ACAPN4-11 6.5 ± 0.08A 1.79 ± 0.21A 94.5 ± 1.0A 98.83 ± 2.51A 93.33 ± 1.52A

Same capital letters indicate non-significant difference estimated by Duncan’s test (P ≤ 0.01) for all tests.a Mycelial growth was measured after incubation on CM medium at 25–28 ◦C for 9 days.

al suspg a toted am

icsT4tp

3

tsf

b Conidiation was measured by counting the number of conidia in a 10 �L conidic Conidial germination was measured by counting the germinated conidia amond Percent appressorial formation was analyzed by counting the appressoria form

n other eukaryotic organisms (Fig. S1). MoCAPN1 and MoCAPN3ontain the CysPc motif, that is the calpain-like cysteine proteaseequence motif. MoCAPN4 contains EF-hand domain (EFh) (Fig. 1).he MoCAPN1, MoCAPN3 and MoCAPN4 proteins show 34, 35 and5% sequence identity to the protein of Homo sapiens, 62, 63, 71%o the protein of Gaeumannomyces graminis, 44, 49 and 48% to therotein of Aspergillus niger.

.2. Disruption of MoCAPN1, MoCAPN3 and MoCAPN4


Three different (5.4, 3.5 and 3.8-kb) gene disruption vec-ors (pBS-up-Sur-dn, pBS-up-Neo-dn and pBS-Hph-dn) containingulfonylurea (Sur), neomycin (Neo) and hygromycin phosphotrans-erase (hph) genes as markers were generated to find the function

Fig. 4. Mycelium growth assays for Guy-11 and mutants on

ension using hemacytometer under a microscope.al of 200 conidia in a 20 �L conidial suspension grown at 25–28 ◦C for 2 and 4 h.ong a total of 200 conidia in a 20 �L conidial suspension grown at 25–28 ◦C for 6 h.

of MoCAPN1, MoCAPN3 and MoCAPN4 genes in rice blast disease(Fig. 2). The disruption vectors were then transformed to theGuy-11 conidia using AGL-1 strain of A. tumefaciens. Chlorimuron,geneticin and hygromycin-resistant transformants after screenedon DCM and CM media were initially confirmed by PCR using Checkprimers (CAPN1-Check-P1: 5′-TGCGTGGGAATAGGTTGTCCTCAA-3′

and CAPN1-Check P2: 5′-GCCAAGGCTCCAAACATCAACCTT-3′,CAPN3-Check-P1: 5′-TGCGTGGGAATAGGTTGTCCTCAA-3′ andCAPN3-Check P2: 5′-GCCAAGGCTCCAAACATCAACCTT-3′ andCAPN4-Check-P1: 5′-TGCGTGGGAATAGGTTGTCCTCAA-3′ and


CAPN4-Check P2: 5′-GCCAAGGCTCCAAACATCAACCTT-3′) result-ing in amplification of 415, 342 and 551-bp fragments. Finallytwo transformants for each gene were confirmed by southernblot analysis to be deleted mutants by homologous recombination

MM, MM-C and MM-N media at 25–28 ◦C for 7 days.




F oculatC

(idkp(AgCACmaM

Ft

ig. 5. Effect of genes disruption on cell wall integrity. Guy-11 and mutants were inolony diameters were measured 7 days post-incubation and photographed.

Fig. 2). The transformants were then purified by mono-conidialsolation and further analyzed for phenotypic studies. Using then-fragment (1.5-kb), up-fragment (1.0-kb) and dn-fragment (1.2-b) of MoCAPN1, MoCAPN3 and MoCAPN4 genes respectively as arobes, obtained by PCR from genomic DNA of Guy-11 with primersdn-P1: 5′-AAggatccGTGCGCCCGAGGTAGCTTTTCTTG-3′, dn-P2: 5′-AtctagaCACGCGCCTTATCTATGGGGACAG-3′), (up-P1: 5′-GTctcgaGAAGACGCGAGGAGGAAGGTTA-3′, up-P2: 5′-GTgatatcCGCGTATGGCACTGGTTGT-3′) and (dn-P1: 5′-GtaagcttAAACGCCAGCCAAGCCAATCAGG-3′, dn-P2: 5′-GtcccgggCGAAACACTACCGCGGGAGAACA-3′) and Sal1, EcoRV and Stu1 as enzymes, the deleted


utants showed 7.3, 4.0 and 2.8-kb bands compared to 2.4, 7.0nd 1.7-kb bands in the Guy-11 strain in MoCAPN1, MoCAPN3 andoCAPN4 genes respectively (Fig. 2).

ig. 6. Stress response of the mutants on MM medium containing different chemicals (Cheir colony diameters were measured and photographed.

ed on CM medium containing Congo red dye at a final concentration of 200 �g/mL.

3.3. Assay for mycelium radial growth and conidiation

For mycelium radial growth analysis, Guy-11 and mutants weregrown on CM medium at 25–28 ◦C for 9 days and diameter ofmycelium growth (cm) was recorded. The colony diameter of themutants of all the deleted genes was similar to that of the Guy-11(Fig. 3A–C). These results showing that these genes are not requiredfor culture colony growth.

As Futai et al. (1999) has reported the role of calpains in coni-dia production in S. cerevisiae, we also analyzed the deleted strainsof calpains-related genes for sporulation by counting the number


of conidia in a 10 �L drop of conidial suspension using hemacy-tometer. Conidia production by the mutants was similar to that ofthe Guy-11 strain. However, mutants for MoCAPN1 gene displayed

uSO4, ZnSO4 and CdCl2). Fungal strains were incubated at 25–28 ◦C for 7 days and




F henotyN

stTwMCsMs

3

1a

TR

S(o

MM, MM-C and MM-N media at 25–28 ◦C for 7 days and diameter

ig. 7. Effect of genes disruption on osmotic (NaCl) and oxidative stress (H2O2). PaCl (0.5 M) were photographed after incubation on CM at 25–28 ◦C for 7 days.

ignificantly reduced (0.57 ± 0.15 and 0.54 ± 0.05) conidia produc-ion compared to that (1.69 ± 0.13) in the Guy-11 strain (Table 1).o ensure that the defect of conidiation in mutants of MoCAPN1as associated with the gene replacement events. The full lengthoCAPN1 gene was reintroduced to the mutants (CAPN1-16 and

APN1-35). Conidia production by the mutants were recovered andimilar to that of the Guy-11 strain (Fig. S2). We concluded thatoCAPN1 protein of this pathogenic fungus may also serve as a

ignal necessary for conidia formation.

.4. Assay for conidial germination and appressorium formation


Conidial germination and appressorial formation for the Guy-1 and mutants of MoCAPN1, MoCAPN3 and MoCAPN4 genes werenalyzed by incubating a drop (20 �L each) of conidial suspension

able 2adial growth (cm) of the mutants and Guy-11 on different media.

Strains CM CM-NaCl CM-CR CM-H2O2

Guy-11 3.8 ± 0.0A 3.46 ± 0.05A 3.63 ± 0.15A 3.13 ± 0.05ACAPN1-16 3.96 ± 0.05A 3.5 ± 0.1A 3.76 ± 0.11A 3.06 ± 0.05ACAPN1-35 3.86 ± 0.05A 3.43 ± 0.05A 3.8 ± 0A 3.2 ± 0ACAPN3-4 3.86 ± 0.05A 3.56 ± 0.11A 3.73 ± 0.05A 3.2 ± 0.1ACAPN3-7 3.93 ± 0.05A 3.53 ± 0.05A 3.76 ± 0.11A 3.16 ± 0.05ACAPN4-9 3.76 ± 0.15A 3.43 ± 0.05A 3.73 ± 0.05A 3.13 ± 0.15ACAPN4-11 3.83 ± 0.05A 3.4 ± 0A 3.66 ± 0.05A 3.13 ± 0.11A

ame capital letters indicate non-significant difference estimated by Duncan’s testP ≤ 0.01) for all tests. Mycelial growth was recorded after incubation of the strainsn CM, CM-NaCl, CM-CR and CM-H2O2 at 25–28 ◦C for 7 days.

pes of Guy-11 and disrupted mutants on CM media containing H2O2 (5 mM) and

(1 × 105 conidia/mL) on sterile plastic coverslips at 25–28 ◦C for2, 4 and 6 h using hemacytometer. Germ tubes and appressoriaformation were similar among the mutants and Guy-11 (Table 1)suggesting no role in these developmental processes.

3.5. Mutants of MoCAPN1, MoCAPN3 and MoCAPN4 showednormal growth on MM, MM-C and MM-N medium

To find the effect of nitrogen and glucose deficiency on thegrowth of deleted mutants, the Guy-11 and mutants were grown on


of mycelium radial growth (cm) was recorded. The mutants werefound able to efficiently utilize the carbon and nitrogen sourcesand grew normally as that of the Guy-11 strain showing that these

Table 3Growth (cm) of the strains of M. oryzae on MM medium containing differentchemicals.

Strains MM MM-CuSO4 MM-ZnSO4 MM-CdCl2

Guy-11 2.66 ± 0.11A 1.6 ± 0.1A 1.1 ± 0A 0.96 ± 0.05ACAPN1-16 2.66 ± 0.05A 1.43 ± 0.11A 1.03 ± 0.05A 0.9 ± 0ACAPN1-35 2.6 ± 0.1A 1.46 ± 0.15A 1.03 ± 0.05A 0.86 ± 0.05ACAPN3-4 2.6 ± 0.1A 1.5 ± 0.26A 1 ± 0A 0.9 ± 0ACAPN3-7 2.66 ± 0.05A 1.56 ± 0.28A 1 ± 0A 0.9 ± 0ACAPN4-9 2.63 ± 0.15A 1.46 ± 0.05A 1 ± 07A 0.86 ± 0.05ACAPN4-11 2.56 ± 0.15A 1.53 ± 0.15A 1.06 ± 0.05A 0.86 ± 0.05A

Same capital letters indicate non-significant difference estimated by Duncan’s test(P ≤ 0.01) for all tests. Mycelial radial growth was measure after culture of all thestrains on MM containing CuSO4, ZnSO4 and CdCl2 at 25–28 ◦C for 7 days.




Table 4Mycelium radial growth (cm) of the mutants on MM containing carbbon and nitro-gen sources.

Strains MM MM-N MM-C

Guy-11 2.66 ± 0.11A 3.3 ± 0.05A 2.93 ± 0.05ACAPN1-16 2.66 ± 0.05A 3.3 ± 0.1A 2.8 ± 0.1ACAPN1-35 2.6 ± 0.1A 3.26 ± 0.05A 2.8 ± 0.1ACAPN3-4 2.6 ± 0.1A 3.16 ± 0.05A 2.83 ± 0.05ACAPN3-7 2.66 ± 0.05A 3.1 ± 0.1A 2.86 ± 0.05ACAPN4-9 2.63 ± 0.15A 3.26 ± 0.11A 2.86 ± 0.05ACAPN4-11 2.56 ± 0.15A 3.2 ± 0.1A 2.83 ± 0.05A

Same capital letters indicate non-significant difference estimated by Duncan’s test(P ≤ 0.01) for all tests.Gm

gT

3i

s1(aTi(owan

3s

MauwNm

3h

gadgcsr

3

fad((

Fig. 8. Infection assay for deleted strains of M. oryzae. (A) Rice leaves inoculatedwith conidial suspension and (B) barley leaves inoculated with mycelium blocks of

rowth was measured after incubation of all the strains on MM, MM-N and MM-Cedia at 25–28 ◦C for 7 days.

enes are not involved in glucose and nitrogen utilization (Fig. 4,able 3).

.6. MoCAPN1, MoCAPN3 and MoCAPN4 play no role in cell wallntegrity

Defects in cell wall composition in M. oryzae can affect appres-orium formation and successful infection of rice plant (Xu et al.,998 and 2000; Skamnioti et al., 2007; Jeon et al., 2008). Congo RedCR), which binds to cell wall component beta-1,4-glucan (Woodnd Fulcher, 1983), is commonly used to detect cell wall integrity.o investigate the function of calpains-related genes in cell wallntegrity, all strains were grown on CM medium amended with CR200 �g/ml) for 7 days. We found that the mutants grew normallyn CM-CR medium showing non-significant involvement in cellall integrity. Halo degradation of CR was similar in both mutants

nd wild-type strains (Fig. 5, Table 2) showing that the mutants areot deficient in CR-degrading activity.

.7. MoCAPN1, MoCAPN3 and MoCAPN4 are not involved intress response under chemicals pressure

Wild-type strain Guy-11 and mutants were grown on solidM medium supplemented with ZnSO4 (10 mM), CuSO4 (1 mM),

nd CdCl2 (0.5 mM) followed by incubation at 25–28 ◦C for 7 daysnder dark conditions. Diameters of representative fungal coloniesere measured and photographed 7 days post-inoculation (Fig. 6).o significant difference was found between the growth of theutants and Guy-11 strain (Table 4).

.8. MoCAPN1, MoCAPN3 and MoCAPN4 play no role inypersensitivity to osmotic and oxidative stress

To investigate the hypersensitive function of calpains-relatedenes, all the Guy-11 and mutants were grown on CM mediummended with H2O2 (5 mM) and NaCl (0.5 M) at 25–28 ◦C for 7ays under dark conditions. The mutants showed normal myceliarowth similar to that of the Guy-11 (Table 2, Fig. 7). This indi-ates that the mutants are resistant to the oxidative and osmotictress suggesting that these genes play no role in H2O2 and NaClesistance.

.9. Pathogenicity test

M. oryzae causes disease by using its own stored energy obtainedrom carbohydrate degradation in conidial cell with the help of


penetration peg emerged from appressorium after conidial celleath. For rice pathogenicity, 14-day-old blast susceptible riceCO-39) seedlings were inoculated with 10 mL conidial suspension5 × 104 conidia/mL) of Guy-11 and mutants. The mutants caused

Guy-11 and mutants were photographed 7 and 4 days post-inoculation respectively.

similar blast lesions as those of the wild-type strain Guy-11 (Fig. 8).7-day-old blast susceptible barley cultivar (zj-8) was also inocu-lated with the 5 mm mycelium blocks of the Guy-11 and mutants.The mutants did not lose its ability to cause pathogenicity andcaused necrotic, proliferating and coalesced blast lesions similarto those of the Guy-11 strain (Fig. 8). We hypothesize that full vir-ulence of the fungus might be correlated with the statement thatdisrupting calpains has no effect on germ tubes and appressoriaformation and hence fully development of the intracellular pro-cesses such as germ tubes and appressoria formation and conidialcell death might result in full virulence of the fungus.

3.10. MoCAPN1, MoCAPN3 and MoCAPN4 play no role in sexualreproduction

In order to determine the role of these genes in sexual reproduc-tion, the wild-type Guy-11 and deleted mutants were crossed withopposite mating type 2539 isolate of M. oryzae to allow peritheciaproduction. Agar plug inocula of the strains were placed on oatmealagar medium and incubated under constant fluorescent light at22 ◦C for 28 days. After 4 weeks of incubation, we observed numer-ous perithecia at the junctions between mated individuals (Fig. 9)


indicating that these genes are not essential for sexual reproductionin M. oryzae.




F cia pr

4

pmp2Ahk

autpcTclcdnow

pc(dditahWdtpnia(cTi

mgtuo

ig. 9. Mating of the mutants and Guy-11 with 2539 strain. Strains showed perithe

. Discussion

Calpains are a family of calcium-activated intracellular cysteineroteases that play important roles in cell motility growth coneotility and guidance (Robles et al., 2003), cell proliferation, apo-

tosis, memory and learning (Aszodi et al., 1991; Battaglia et al.,003; Glading et al., 2002; Gil-Parrado et al., 2002; Wang, 2000).lthough calpains have been reported to play important roles inuman pathogenesis (Huang and Wang, 2001), however, little isnown about the functions of calpains in filamentous fungi.

In this study we characterized three calpains-related genesnd finally analyzed their function in infection-related event bysing mutants throughout our whole study for conidiation, cul-ure colony growth, conidial germination, appressorium formation,athogenicity, osmotic and oxidative stress response, mating test,ell wall integrity test and growth on MM, MM-C and MM-N media.o find the role of these calpains in blast-related events, we firstloned these calpains and then disrupted them for mutants iso-ation through gene disruption method. Gene disruption of thesealpains produced mutants with usual growth characteristics, coni-ia production, germination, appressorium formation, displayedormal growth on glucose and nitrogen deficient media, un-alteredsmotic and oxidative response, showed perithecia during matingith opposite mating type 2539 strain and full pathogenicity.

Finally, we also determined the roles of these calpains forathogenicity and sexual reproduction. We inoculated the sus-eptible rice and barley seedlings with 10 mL conidial suspension5 × 104 conidia/mL) and mycelium blocks (5 mm) of Guy-11 andeleted mutants and incubated the inoculated seedlings for 5 and 4ays respectively. We found that the deleted mutants caused sim-

lar blast lesions showing coalesced, proliferating and necrotic ashose of the Guy-11 strain. This result indicates that these calpainsre not required for the blast fungus to cause pathogenicity andence these genes are pathogenicity non-essential genes (Fig. 8).e also investigated the roles of these calpains in sexual repro-

uction by crossing the deleted strains with the opposite matingype 2539 strain and found that the mutants produced numerouserithecia 28 days post incubation suggesting that these genes areot essential for sexual reproduction in M. oryzae (Fig. 9). Conid-

ogenesis was also determined by growing all the deleted strainnd Guy-11 on complete medium for 9 days showing reduction0.57 ± 15.65B and 0.54 ± 5.15B) in the mutants for MoCAPN1 geneompared to that (1.69 ± 13.21A) in the Guy-11 strain (Table 1).his result has also been supported by the studies on calpains rolen conidiation in S. cerevisiae (Futai et al., 1999).

Conidial germination and appressorial formation for all theutants and Guy-11 strain were almost similar showing that these


enes are not critical for conidia production and appressoria forma-ion (Table 1). Cell wall integrity (congo red) test, stress responsender chemical pressure (ZnSO4, CuSO4 and CdCl2), osmotic andxidative (NaCl and H2O2) stress response and growth on CM

oduction suggesting that these genes are not essential for sexual reproduction.

media at 9 dpi resulted in similar results as those of the Guy-11(Figs. 2, 4, 5, 6 and Tables 2–4). We also analyzed the growthresponse of the mutants on glucose and nitrogen deficient mediaas the absence of nitrogen source combined with the presence ofa carbon source leads cells to enter the developmental pathway ofsporulation (Freese et al. 1982). The mutants grew normally similarto those of the Guy-11 (Fig. 4).

We found seven calpains-related genes in M. oryzae by align-ment as shown in Fig. S1. MoCAPN1, 3 and 4 could be deletedrespectively using target-gene replacement method and themutants showed major phenotypes similar to those of the Guy-11 strain. However, the rest of the calpains-related genes (shownin Fig. S1) could not be disrupted successfully using this methodalthough hundreds of transformants were checked. It is reportedthat defects in ubiquitous calpains may be lethal (Tan et al., 2006;Kashiwagi et al., 2010). Therefore, calpains might play importantrole in M. oryzae. The full function of the calpains-related genescould not detected using target-gene replacement method. It isnecessary to address the molecular mechanism of calpains in M.oryzae further. How the calpains regulating the sporulation path-way needs to be further elucidated and this might shed further lighton the biology of calpains in this fungus.

Acknowledgements

This work was supported by grants (no. 31370171 and31371890) funded by the National Natural Science Foundation ofChina, and our staff and graduate students at the Fungal Laboratory,Biotechnology Institute, Zhejiang University.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.micres.2014.03.003.

References

Adachi K, Hamer JE. Divergent cAMP signaling pathways regulate growth and patho-genesis in the rice blast fungus Magnaporthe grisea. Plant Cell 1998;10:1361–73.

Aszodi A, Muller U, Friedrich P, Spatz HC. Signal convergence on protein kinase A asa molecular correlate of learning. Proc Natl Acad Sci USA 1991;88(13):5832–6.

Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP. Signaling in plant–microbeinteractions. Science 1997;276:726–33.

Battaglia F, Trinchese F, Liu S, Walter S, Nixon RA, Arancio O. Calpaininhibitors, a treatment for Alzheimer’s disease: position paper. J Mol Neurosci2003;20(3):357–62.

Choi W, Dean RA. The adenylate cyclase gene MAC1 of Magnaporthe grisea controlsappressorium formation and other aspects of growth and development. Plant


Cell 1997;9:1973–83.Clergeot PH, Gourgues M, Cots J, Laurans F, Latorse MP, Pepin R, et al. PLS1, a

gene encoding a tetraspanin-like protein, is required for penetration of riceleaf by the fungal pathogen Magnaporthe grisea. Proc Natl Acad Sci U S A2001;98(12):6963–8.


http://dx.doi.org/10.1016/j.micres.2014.03.003

http://dx.doi.org/10.1016/j.micres.2014.03.003

http://refhub.elsevier.com/S0944-5013(14)00043-3/sbref0005


























































































































ING ModelM

ical R

C

D

D

F

F

F

G

G

G

G

G

H

H

J

K

K

K

K

L

L

L

L

L

L

L

L

L

L



roall DE, Ersfeld K. The calpains: modular designs and functional diversity. GenomeBiol 2007;8:218.

eZwaan TM, Carroll AM, Valent B, Sweigard JA. Magnaporthe grisea Pth11p is anovel plasma membrane protein that mediates appressorium differentiation inresponse to inductive substrate cues. Plant Cell 1999;11(10):2013–30.

ou X, Wang Q, Qi Z, Song W, Wang W, Guo M, et al. MoVam7, a conservedSNARE involved in vacuole assembly, is required for growth, endocytosis, ROSaccumulation, and pathogenesis of Magnaporthe oryzae. PLoS ONE 2011;6(1):e16439.

ranco SJ, Huttenlocher A. Regulating cell migration: calpains make the cut. J CellSci 2005;118:3829–38.

reese EB, Chu MI, Freese E. Initiation of yeast sporulation of partial carbon, nitrogen,or phosphate deprivation. J Bacteriol 1982;149:840–51.

utai E, Maeda T, Sorimachi H, Kitamoto K, Ishiura S, Suzuki K. The proteaseactivity of a calpain-like cysteine protease in Saccharomyces cerevisiae isrequired for alkaline adaptation and sporulation. Mol Gen Genet 1999;260:559–68.

eisen R. Cloning of a protease gene from Pseudomonas nalgiovense by expressionin Escherichia coli. Lett Appl Microbiol 1993;16:303–6.

il-Parrado S, Fernandez-Montalvan A, Assfalg-Machleidt I, Popp O, Bestvater F, Hol-loschi A, et al. Ionomycin-activated calpain triggers apoptosis. A probable rolefor Bcl-2 family members, J Biol Chem 2002;27(30):27217–26.

lading A, Lauffenburger DA, Wells A. Cutting to the chase: calpain proteases in cellmotility. Trends Cell Biol 2002;12(1):46–54.

oll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev2003;83:731–801.

uroff G. A neutral, calcium-activated proteinase from the soluble fraction of ratbrain. J Biol Chem 1964;239:149–55.

oward RJ, Valent B. Breaking and entering: host penetration by the fungal rice blastpathogen Magnaporthe grisea. Annu Rev Microbiol 1996;50:491–512.

uang Y, Wang KK. The calpain family and human disease. Trends Mol Med2001;7:355–62.

eon J, Goh J, Yoo S, Chi MH, Choi J, Rho HS, et al. A putative MAP kinase kinasekinase, MCK1, is required for cell wall integrity and pathogenicity of the riceblast fungus. Magnaporthe oryzae, MPMI 2008;21(5):525–34.

ang SH, Khang CH, Lee YH. Regulation of cAMP dependent protein kinaseduring appressorium formation in Magnaporthe grisea. FEMS Microbiol Lett1999;170:419–23.

ashiwagi A, Schipani E, Fein MJ, Greer PA, Shimada M. Targeted deletion of Capn4in cells of the chondrocyte lineage impairs chondrocyte proliferation and differ-entiation. Mol Cell Biol 2010;30:2799–810.

im S, Ahn IP, Rho HS, Lee YH. MHP1, a Magnaporthe grisea hydrophobin gene,is required for fungal development and plant colonization. Mol Microbiol2005;57:1224–37.

ornberg A, Spudich JA, Nelson DL, Deutscher MP. Origin of proteins in sporulation.Annu Rev Biochem 1968;37:51–78.

ebart MC, Benyamin Y. Calpain involvement in the remodeling of cytoskeletalanchorage complexes. FEBS J 2006;273:3415–26.

ee YH, Dean RA. cAMP regulates infection structure formation in the plantpathogenic fungus Magnaporthe grisea. Plant Cell 1993;5:693–700.

i HJ, Lu JP, Liu XH, Zhang LL, Lin FC. Vectors Building and Usage for Gene Knockout,Protein Expression and Fluorescent Fusion Protein in The Rice Blast Fungus. JAgric Biotechnol 2011;20(1):94–104.

i Y, Liang S, Yan X, Wang H, Li D, Soanes DM, et al. Characterizationof MoLDB1 required for vegetative growth, infection-related morphogene-sis, and pathogenicity in the rice blast fungus Magnaporthe oryzae. MPMI2010;23:1260–74.

iu XH, Lu JP, Lin FC. Autophagy during conidiation, conidial germination and turgorgeneration in Magnaporthe grisea. Autophagy 2007:3.

iu XH, Liu TB, Lin FC, et al. Monitoring autophagy in Magnaporthe oryzae. MethodsEnzymol 2008;451:271–94.

iu TB, Chen GQ, Min H, Lin FC. MoFLP1, encoding a novel fungal fasciclin-like protein,is involved in conidiation and pathogenicity in Magnaporthe oryzae. J ZhejiangUniv Sci B 2009;10:434–44.

iu XH, Yang J, He RL, Lu JP, Zhang CL, Lu SL, et al. An autophagy gene,TrATG5, affects conidiospore differentiation in Trichoderma reesei. ResMicrobiol 2011;162(8):756–63.


iu S, Dean RA. G protein a subunit genes control growth, development andpathogenicity of Magnaporthe grisea. MPMI 1997;10(9):1075–86.

u JP, Feng XX, Liu XH, Lu Q, Wang HK, Lin FC. Mnh6, a nonhistone protein, is requiredfor fungal development and pathogenicity of Magnaporthe grisea. Fungal GenetBiol 2007;44(9):819–29.

PRESSesearch xxx (2014) xxx–xxx 11

Mamoune A, Luo JH, Lauffenburger DA, Wells A. Calpain-2 as a target for limitingprostate cancer invasion. Cancer Res 2003;63:4632–40.

Mitchell TK, Dean RA. The cAMP-dependent protein kinase catalytic subunitis required for appressorium formation and pathogenesis by the rice blastpathogen Magnaporthe grisea. Plant Cell 1995;7:1869–78.

North MJ. Comparative biochemistry of the proteinases of eukaryotic microorgan-isms. Microbiol Rev 1982;46:308–40.

Odenbach D, Breth B, Thines E, Weber RW, Anke H, Foster AJ. The transcription factorCon7p is a central regulator of infection-related morphogenesis in the rice blastfungus Magnaporthe grisea. Mol Microbiol 2007;64:293–307.

Ou SH. Rice diseases. 2nd ed. UK: Commonwealth Agricultural Bureaux; 1985. pp.380.

Park G, Xue C, Zhao X, Kim Y, Orbach M, Xu JR. Multiple upstream signals converge onan adaptor protein Mst50 to activate the PMK1 pathway in Magnaporthe grisea.Plant Cell 2006;18:2822–35.

Phadatare S, Rao M, Deshpande V. A serine alkaline protease from the fungus Coni-diobolus coronatus with a distinctly different structure than the serine proteasesubtilisin Carlsberg. Arch Microbiol 1997;166:414–7.

Robles E, Huttenlocher A, Gomez TM. Filopodial calcium transients regulategrowth cone motility and guidance through local activation of calpain. Neuron2003;38(4):597–609.

Sambrook J, Russell DW. Molecular cloning: a laboratory manual, vols. 1, 2, 3, 3rded. New York, NY: Cold Spring Harbor Laboratory Press; 2002.

Shiba E, Kambayashi JI, Sakon M, Kawasaki T, Kobayashi T, Koyama H, et al.Ca2+-dependent neutral protease (calpain) activity in breast cancer tissue andestrogen receptor status. Breast Cancer 1996;3:13–7.

Skamnioti P, Gurr SJ. A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea.MPMI 2007;20(5):568–80.

Sorimachi H, Ishiura S, Suzuki K. Structure and physiological function of calpains.Biochem J 1997;328:721–32.

Sweigard JA, Chumley FG, Valent B. Disruption of a Magnaporthe grisea cutinase gene.Mol Gen Genet 1992;232(2):183–90.

Talbot NJ, Ebbole DJ, Hamer JE. Identification and characterization of MPG1 a geneinvolved in pathogenicity from the rice blast fungus Magnaporthe oryzae. PlantCell 1993;5(11):1575–90.

Tan Y, Dourdin N, Wu C, De Veyra T, Elce JS, Greer PA. Conditional disruption ofubiquitous calpains in the mouse. Genesis 2006;44:297–303.

Thines E, Weber RW, Talbot NJ. MAP kinase and protein kinase A-dependent mobi-lization of triacylglycerol and glycogen during appressorium turgor generationby Magnaporthe grisea. Plant Cell 2000;12:1703–18.

Tucker SL, Thornton CR, Tasker K, Jacob C, Giles G, Egan M, et al. A fungalmetallothionein is required for pathogenicity of Magnaporthe grisea. Plant Cell2004;16:1575–88.

Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot NJ. Autophagic fungal celldeath is necessary for infection by the rice blast fungus. Science 2006;312:580–3.

Veneault-Fourrey C, Talbot NJ. Moving toward a systems biology approach to thestudy of fungal pathogenesis in the rice blast fungus Magnaporthe grisea. AdvAppl Microbiol 2005;57:177–215.

Wang KK. Calpain and caspase: can you tell the difference? Trends Neurosci2000;23(1):20–6.

Wood PJ, Fulcher RG. Dye interactions, a basis for specific detection and histoche-mistry of polysaccharides. Histochemistry Cytochemistry 1983;31(6):823–6.

Wu SC, Halley JE, Luttig C, Fernekes LM, Gutierrez-Sanchez G, Darvill AG, et al. Iden-tification of a Magnaporthe grisea endo-beta-1,4-D-xylanase by gene knockoutanalysis, protein purification and heterologous expression. Appl Environ Micro-biol 2006;72:986–93.

Xu. Map kinases in fungal pathogens. Fungal Genet Biol 2000;31(3):137–52.Xu JR, Staiger CJ, Hamer JE. Inactivation of the mitogen-activated protein kinase

mps1 from the rice blast fungus prevents penetration of host cells but allowsactivation of plant defense responses. Proc Natl Acad Sci U S A 1998;95:12713–8.

Xu JR. MAP kinases in fungal pathogens. Fungal Genet Biol 2000;31:137–52.Zeigler RH, Leong SA, Teong PS. Rice blast disease. Los Banos: CAB International;

1994, 626 pp.Zhang H, Li K, Zhan X, Tan W, Wan J, Gu M, et al. Two phosphodiesterase genes, PDEL

and PDEH, regulate development and pathogenicity by modulating intracellularcyclic AMP levels in Magnaporthe oryzae. PLoS ONE 2011;6(2):e17241.


Zhao X, Xu JR. A highly conserved MAPK-docking site in Mst7 is essential for Pmk1activation in Magnaporthe grisea. Mol Microbiol 2007;63:881–94.

Zhao X, Kim Y, Park G, Xu JR. A mitogen-activated protein kinase cascade reg-ulating infection-related morphogenesis in Magnaporthe grisea. Plant Cell2005;17:1317–29.





























































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































Documents

Disruption and molecular characterization of calpains-related (MoCAPN1, MoCAPN3 and MoCAPN4) genes in Magnaporthe oryzae