A ‘genome to paddock’ approach

to control plant disease

Barbara Howlett, School of BioSciences

the University of Melbourne

Previous Research Projects

• B.Sc (Hons) Cytochrome C (Melb Uni)

• Research Assistant: Tapeworms (CSIRO, Melb)

• Research Assistant: Influenza (ANU)

• M.Sc: Self incompatibility in plants (ANU)

• Research Assistant: Bacterial chemotaxis (UC Berkeley)

• Ph.D: Grass pollen allergens (Melb Uni)

• Post doc: Rhizobia and nitrogen fixation (Stanford)

• Research Fellow: Phytophthora vignae – cowpea interaction (Melb Uni)

Neurospora crassa: model fungus

Namboori RajuAsci from cross between isolate Histone H1- GFP X wild type

Separating fungal chromosomes on gels

Mb

BioRad CHEF Prototype Saccharomyces cerevisiae

Blackleg caused by Leptosphaeria maculans

• Canola industry established in 1970s but severe blackleg epidemics

• Resistance breeding programs

• Industry re-established in 1990s

• Ascomycete fungus

• Grows on defined media

• Reproducible inoculation protocols

• Genetics: sexual crossing

Canola and blackleg disease in Australia

stubble sexual fruiting bodies windborne ascospores

stem

canker

cotyledon infectionroot rotlodged

plants

SUMMER AUTUMN

SPRING WINTER

Canola in Australia: minimum tillage, long

growing season, temperate climate, high

intensity cropping

2.5 m ha canola sown in 2016; large amount of

blackleg-infested stubble inoculum in 2017

Controlling Blackleg Disease

• Rotation with cereals & pulses

• Fungicide applied to seed, ‘in furrow’ and/or spray

• Resistance breeding; seedling (major gene) & adult plant (minor gene) resistance

No resistance

= disease

Adult (minor gene)

resistance

= some disease

Seedling (major

gene) resistance

= no disease

‘Gene for gene’ interaction

• >10 major resistance genes mapped (LepR3, Rlm2 cloned)

• Avirulence genes cloned (Rouxel INRA, Van de Wouw, Melb Uni)

• Typical effectors: small secreted cysteine-rich proteins

Avirulence gene Resistance gene

AvrLm1 Rlm1

AvrLm6 Rlm6

AvrLm4-7 Rlm4 & Rlm7

AvrLm11 Rlm11

AvrLm5 Rlm5

AvrLm2 Rlm2

AvrLm3 Rlm3

AvrLepR1 LepR1

AvrLepR2 LepR2

AvrLepR3 LepR3

‘Breakdown’ of resistance in 2003

• in 2000 ‘Sylvestris’ cultivars released with resistance gene, LepR3

• Sown extensively on Eyre Peninsula, SA

Eyre Peninsula

2001 and 2002 2003

‘Sylvestris’ cultivars (LepR3)

Beacon

90% yield losses ($30 m) in Eyre Peninsula

Seed withdrawn from sale

2003: Sylvestris

Weekly TimesThe Land

2004: Beacon Sylvestris

2002 2003 2006

Sylvestris resistance

2002 2003 2006

Non-Sylvestris resistance

• Prolific asexual & sexual reproduction - recombination. Large populations of windborne ascospores (inoculum)

• Increased frequency of isolates attacking cultivars with Sylvestris-resistance, not new strain:– isolates collected before 1988 caused disease– breakdown other areas where Sylvestris cultivars sown extensively

• Extensive sowing of cultivars with major gene resistance results in strong selection pressure towards isolates virulent to that resistance gene. Frequency of virulent isolates in populations increases

• What features of the fungal genome allow virulence to evolve so rapidly in field populations?

• What changes in avirulence genes are responsible for breakdown of Sylvestris resistance?

Leptosphaeria maculans - high risk

pathogen for ‘overcoming’ resistance

Spores land on leaf and germinate

Hyphae grow across leaf and enter stomata

Successful invasion Unsuccessful invasion

Chen and Howlett (1996) Physiol. Mol. Plant Path. 48: 73-81

Successful invasion

leaf

stem

root

Hyphae (green) grow in leaf into stem then into root Sprague, Watt, Kirkegaard, Howlett (2007) New Phytol 176: 211-22.

Manipulating the blackleg fungus in the

lab

Kim Plummer

Robin Williams

Genome structure: isolates have different-sized

chromosomes

# bands (chromosomes) =16

add sizes of each band (genome size) = 38 Mb

Plummer and Howlett (1993) Curr Genet 24: 107-13

Progeny can have different sized homologous

chromosomes to those of parents

P1 P3P2 P4 P1 P3P2 P4

Same size as parents

Different size to parents

progeny progeny progeny progeny

Plummer and Howlett (1995) Mol Gen Genet 247: 416-22

Chromosomal length polymorphisms due to different amounts of repetitive DNA in homologs

Slippage (unequal pairing) during meiosis can lead to generation of length polymorphisms

Repetitive DNA is transposon-derived

Inheritance of chromosomal length polymorphisms

Mutation: Repeat Induced Point (RIP)

• Genome defence against invasion of fungi by transposable

elements?

• Mutates multicopy DNA; occurs in haploid nuclei after

fertilization, prior to mitotic DNA replication in N.crassa

• Transitions from C:G to T:A base pairs; often generates

stop codons

• RIP ‘signature’ deduced by bioinformatics

• Demonstrated experimentally in L.maculans (Alex Idnurm)

• Idnurm & Howlett (2003) Curr Gen 39: 31-7

Leptosphaeria maculans genome sequencing

• Rouxel & Balesdent, INRA & Howlett, UoM;

Genoscope; URGI, France; Oliver (Perth)

• 12,500 genes; 45 Mb -closely related Stagonospora

nodorum (37 Mb)

• 36% genome (9% S. nodorum) is AT-rich repetitive

DNA comprising degenerated transposable elements

• Repeat Induced Point (RIP) mutation in repetitive

DNA

• Unique genome structure

‘Patchwork’ genome: gene–rich (GC) & gene-

poor, repeat –rich repetitive (AT) blocks

Gene-rich blocks(1 gene per 2.4 kb)

Repeat-rich blocks

Gene poor (1 gene/ 30 kb) degenerated transposons only 3.5% of total genes but 20% of effectors

Rouxel et al. Nature Communications (2011) 2: 22

ATTATTATTATTATTATTATTATTATTATTATTATTATTATTA

If repeats align correctly during meiosis

Location of avirulence genes in repetitive regions can lead to gene loss or mutation

Avr

or

Avirulence genes identical in parents & progeny

During sexual reproduction, sister

chromatids align

Crossovers can occur (recombination)

ATTATTATTATTATTATTATTATTATTATTATTATTATTATTA

ATTATTATTATTATTATTATTATTATTATTATTATTATTATTA

ATTATTATTATTATTATTATTATTATTATTATTATTATTATTAAvr

Avr

Avr

If repeats align incorrectly(slippage) during meiosis

Location of avirulence genes in repetitive regions can lead to gene loss or mutation

ATTATTATTATTATTATTATTATTATTATTATTA

Deletion of Avirulence gene

ATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTAvr

ATTATTATTATTATTATTATTATTATTATTATTATTATT

ATTATTATTATTATTATTATTATTATTATTATTATTATTATTA

Gene duplication

Avr

Avr Avr

RIP mutation

Resistance breakdown and evolution of

virulence

• Why does virulence evolve so rapidly in the blackleg fungus?

• Avirulence genes are located in repetitive regions of genome where RIP mutation and gene loss occur readily

• Sylvestris cultivars have Rlm1 and LepR3

• What changes in AvrLm1 are responsible for breakdown of Sylvestris resistance?

Genomic environment of AvrLm1

• 530 kb region: repetitive (AT- rich) DNA, recombination-deficient

• AvrLm1, AvrLm6, LmCys1 and LmCys2 effectors

• Sequenced genes in isolates:

• 137 before breakdown of resistance (before 2004)

• 158 after breakdown (2004 and later)

Features of AvrLm1, AvrLm6, LmCys1 & LmCys2 effectors in 295 isolates

Gene Mutation Virulence

mechanism

Frequencies of

virulence alleles

AvrLm1 Deletion, aa

sub

Deletion only Eight fold increase

post- sylvestris

breakdown

AvrLm6 Deletion, RIP,

aa sub

Deletion

(70%), RIP &

aa sub

Six fold increase

post- sylvestris

breakdown

LmCys1 RIP, aa

substitutions

Not

applicable

Not applicable

LmCys2 Deletion Not

applicable

Not applicable

Van de Wouw et al. (2010) PLoS Path 6: e1001180

Breakdown of ‘sylvestris’ resistance

• First study of evolutionary constraints on multiple genes in large number fungal isolates

• Linked effector genes evolve differently: deletions, Repeat-Induced Point mutations & amino acid substitutions

• Degree of RIP mutation in single copy genes is proportional to proximity to repetitive transposable elements - ‘leakage‘

• Frequency of isolates with virulence alleles of AvrLm1 & AvrLm6 increased after extensive sowing of cultivars with ‘sylvestris’ resistance, even though no Rlm6 cultivars had been sown

• Genomic location (low recombination frequency) led to a selective sweep whereby selection at AvrLm1 affected frequency of virulence alleles of linked AvrLm6 (hitchhiking)

34

Implications for disease management

• Selection imposed by wide-spread deployment of resistance gene affects evolution of complementary avirulence gene and closely linked avirulence genes

• Knowledge of fungal genome important

• Maximise effective lifetime of resistance genes as each resistance gene costs >$4 million to commercialise and release to farmers

• Virulence frequencies of fungal populations can be manipulated by sowing cultivars with different resistance genes, thus minimising risk of resistance breakdown (pot, field plot experiments, Eyre Peninsula crops)

• Is monitoring changes in frequency of virulence then selectively withdrawing cultivars at risk of resistance breakdown an effective disease control strategy?

35

National Canola Pathology Project

NSW DPI:

Kurt Lindbeck

UM:

Alex Idnurm

Barbara Howlett

Angela Van de Wouw

DAFWA:

Ravjit Khangura

SARDI:

Andrew Ware

Jenny Davidson

MGP:

Steve Marcroft

Angela Van de Wouw

Elizabeth Sheedy

Alistair Smith

Buffy Harrison

CSIRO:

Susie Sprague

NSW DPI:

Kurt Lindbeck

UM:

Alex Idnurm

Barbara Howlett

Angela Van de Wouw

DAFWA:

Ravjit Khangura

SARDI:

Andrew Ware

Jenny Davidson

CSIRO:

Susie Sprague

‘Genome to Paddock’ holistic approach

Monitoring fungal popsAvr gene identificationFungicide tolerance?

Monitoring field diseaseBlackleg resistance groups

Blackleg Management Guide

Canola

Resistance

Fungal

virulence

Sustainable control

of blackleg disease

MGP:

Steve Marcroft

Elizabeth Sheedy

Angela Van de Wouw

Alistair Smith

Buffy Harrison

• Rotating resistance genes requires knowledge of which

genes are in which cultivars

• Inoculate with 12 ‘differential’ isolates (different Avirulence genes) to classify all breeding lines according to complement of resistance genes

• Resistance groups (A-H) based on complement of resistance genes

Virulent(susceptible reaction)

Avirulent(resistant reaction)

Resistance genotyping (grouping) of Australian breeding lines and cultivars

eastern Australia

Determine risk of resistance breakdownby monitoring changes in frequency of avirulence in

fungal populations

Survey (Internal Stem Infection)

Western Australia

Collect stubble: high throughput

molecular assay of ascospores for

frequency of avirulence

Tools: Isolate collection & molecular markers

• Isolates (>4000) since 2000; stubble from 2007 onwards

• Eight avirulence genes cloned

• AvrLm1 = Rlm1; AvrLm4 = Rlm4; AvrLm3 = Rlm3; AvrLm6 = Rlm6 (these R genes in Aust. cultivars)

• Genetic mechanisms conferring virulence identified

• Deletion of entire gene

• Single nucleotide polymorphism (SNP) - amino acid substitution

• Repeat Induced Point mutations

• Molecular markers developed - applied to single isolates & populations

• Allele frequencies change rapidly over time; presence of resistance genes influences avirulence gene frequencies

Van de Wouw et al. (2010) Plant Pathology; (2013) J App Microbiol; (2017) Crop and Pasture Science

• Stubble placed in wind tunnel, ascospores captured on tape

• DNA extracted and analysed by quantitative PCR for presence/absence of band for AvrLm1, AvrLm6

• Pyrosequencing assay for AvrLm4 based on nucleotide polymorphism at base 358 leading to aa change G120 to R120

• Total number spores estimated by PCR of ribosomal DNA region

• Frequency virulence allele measured; risk resistance breakdown determined in each region

• Farmers advised on cultivar choice

High throughput molecular assay to assess virulence alleles in fungal populations

• Guide updated with blackleg

ratings, cultural practices,

resistance groups of cultivars,

fungicide recommendations

• Regional disease warnings based

on risk resistance breakdown

• Encourages farmers to survey their

crops

• Message - Staying Ahead of

Blackleg

• Canola Pathology Workshops for

researchers, breeders, pathologists

and industry

Canola

Communication

2011: Field and pot trials predicted resistance breakdown of Hyola 50 on Eyre Peninsula in 2012

44

Hyola50 infected from Hyola50 stubble

Hyola50 infected from ATR-Cobbler stubble

Breakdown of Hyola 50 ResistanceFeb 2012

Disease severity of cultivars from different

resistance groups: Eyre Peninsula field plots

0

20

40

60

80

100

Hyola444 ATR-Marlin ATR-Stingray CB-Telfer Thumper TT

Perc

enta

ge inte

rnal in

fection

Cultivar

Group

AGroup

CGroup

B

Group

E

October 2012

Group

D

Hyola 50: 90% disease severity

Eyre Peninsula October 2012

Group DGroup E

Wagga Wagga NSW Oct 2012

Group D

Averting yield losses on Eyre Peninsula in 2012

• 60,000 ha sown to canola in 2012

• Assuming 50% yield losses if 30% of area sown to Group D cultivars (eg. Hyola 50) and $500 /tonne for canola (conservative estimates)

• Benefits

• Farmers were saved losses of $18 million

• Group D cultivars still sown in other regions, so breeding companies very supportive of recommendations to avoid resistance breakdown

• Win-win situation for farmers and companies

Current disease issues

• Upper branch canopy infection -

early sowing

• Stacking of resistance genes: many

cultivars have Group A. Few

cultivars to ‘rotate’

• Industry now reliant on fungicides

(triazoles only)

• High throughput screen with

stubble from several locations

shows isolates with tolerance to

fluquinconazole (seed dressing)

• Knocking out L.maculans genes

by CRISPR

• L.maculans genes, toxins

involved in stem cankering

• Determining mechanisms of RIP

mutation in L.maculans

• Cryptococcus – animal pathogen

• Environmental sensing by fungi

Current Lab Activities (Alex Idnurm)

Summary

• Multidisciplinary research can be effective at solving problems – and is extremely rewarding

• Blackleg fungus can rapidly breakdown disease resistance bred into canola cultivars

• Propensity for resistance breakdown due to location of fungal avirulence genes in repetitive (AT-rich) DNA where mutation and deletion readily occurs

• Monitoring changes in disease severity and virulence of fungal populations, then selectively withdrawing cultivars at risk of resistance breakdown minimisesblackleg disease