Download Cloning a gene in the blackleg fungus, Leptosphaeria maculans

2/26/2014
Cloning a gene in the blackleg
fungus, Leptosphaeria maculans
conferring avirulence towards
Brassica juncea
Resistance and avirulence genes
• Plant contains resistance genes (R genes)
• Pathogen contains avirulence genes (Avr genes)
corresponding to R genes
Fungus Genotype:
AvrRml1 (Avirulent)
Angela Van de Wouw, Rohan Lowe, Candace
Elliott, David Dubois, Barbara Howlett
School of Botany,
The University of Melbourne
Plant Genotype:
Rlm1
Four avirulence genes have been cloned
from L. maculans
Resistance and avirulence genes
• Plant contains resistance genes (R genes)
• Pathogen contains avirulence genes (Avr genes)
corresponding to R genes
•
•
•
•
AvrLm1 = Rlm1 (AV-Garnet)
AvrLm4 = Rlm4 (CB-Telfer)
AvrLm6 = Rlm6 (B. juncea)
AvrLm11 = Rlm11 (B. rapa)
– All located in gene-poor regions of the genome and highly upregulated in planta
Fungus Genotype:
avrRml1 (Virulent)
Plant Genotype:
Rlm1
Plant recognises fungus
- Defence mechanisms
- NO INFECTION
Fungus undetected by plant
INFECTION/DISEASE
Molecular markers can be developed for
avirulence genes
Molecular markers can be used to monitor
changes in allele frequencies in the field
Frequency of virulence allele
Absence/presence of a band
Nucleotide polymorphism (SNP) detection
Pyrosequencing of AvrLm4
A4: C/ GAAATATAACTCCAGGTGCTGAGCTA
C: 4%
G: 96%
250
200
150
100
50
0
E
S
T
C
G
A
T
A
T
A
5
Van de Wouw et al (2010) Plant Pathology 59, 809-818
Van de Wouw et al (2010) PLoS Pathogens 6(11)e1001180
5
Van de Wouw and Howlett (2012) Journal
of Applied Microbiology 113, 1145-1153
Monola76
AvrLm4
AvrLm1
1
2/26/2014
Candidate B. juncea avirulence gene
A/Virulence towards juncea mapped onto
SuperContig7 of L.maculans
Super Contig 7
• Previous work in Howlett lab identified junceaattacking blackleg isolates
– These isolates attack all 92 B. juncea lines screened
– Virulence towards B. juncea segregated as a single gene
– Species-specific a/virulence gene in L. maculans
• Crosses set up between a junceaattacking isolate (IBCN18) and a nonattacking isolate (04P014).
• Both isolates virulent towards Rlm6, a
juncea-resistance gene introgressed
into B. napus.
• 66 progeny were screened for
virulence on juncea and with
molecular markers
• A/virulence mapped to SC7
Purwantara et al (1998)
Candidate gene identified using RNAseq
• RNA-seq data from infected cotyledons (7 dpi) and
in vitro growth used to identify genes highly upregulated in planta.
• Within mapped region on SC7, three SSPs identified
– two had low expression in planta
– one (LemaT070880) up-regulated 5380 fold in planta
57 kb
gene expression in
planta (FPKM)
Marker
MinLm935-2
SSR104
10.6
MinLm1139
AvrLmJ1
12.1
MinLm1377
21.3
MinLm2007
LemaT070880 is candidate avirulence gene
• LemaT070880 encodes a small secreted, cysteine rich protein
located in AT-rich region
• RNA-seq data showed incorrect gene prediction in reference
genome; correct annotation has earlier start codon.
• Gene sequenced from attacking and non-attacking isolates
33 kb
25 genes
7 genes
886 kb
cM
25.8
24.6
27 genes
1102 kb
AvrLmJ1
1500
1000
500
0
Premature stop-codon in junceaattacking isolates
• Stop codon identified in juncea-attacking isolates
• Presence of stop codon shows 100% correlation with
virulence in progeny
Isolates
# of nucleotide
Coding sequence
Phenotype on B.
Allele
(frequency)
changes
change
juncea cultivars
Lema_uP070880.2_0
34 (43%)
N/A
N/A
Avirulent
Lema_uP070880.2_1
18 (23%)
1
K55R
Avirulent
Lema_uP070880.2_2
15 (19%)
1
K55T
Avirulent
Lema_uP070880.2_3
9 (12%)
2
R38L, K55R
Avirulent
Lema_uP070880.2_4
2 (3%)
3
R29Stop, R38L, K55R
Virulent
LemaT070880 confers avirulence towards
Juncea cultivars - I
• Complementation construct made and transformed
into juncea-attacking isolate
B. napus cv.
Westar
B. juncea cv.
Aurea
IBCN18
IBCN18
+AvrLmJ1 #1
2
2/26/2014
LemaT070880 confers avirulence towards
Juncea cultivars - II
Westar
Stoke
9.0
• LemaT070880 (AvrLmJ1) confers avirulence
towards three B. juncea lines tested.
Aurea
Forge
8.0
Average pathogenicity score
Conclusions
– Possibly confers species-specific avirulence
– Corresponding R gene unknown.
7.0
6.0
• Typical characteristics of avirulence genes
5.0
4.0
– Secretion signal, cystiene rich, no homolgy,
located in AT-rich region and highly up-regulated
in planta
3.0
2.0
1.0
0.0
IBCN18
IBCN18
+AvrLmJ1#1
IBCN18
+AvrLmJ1#2
IBCN18
+AvrLmJ1#3
IBCN18
+AvrLmJ1#4
IBCN18
+AvrLmJ1#5
• RNAseq data in combination with mapping
allowed identification of candidate gene
3