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. 2017 Feb;213(3):1301-1314.
doi: 10.1111/nph.14372. Epub 2016 Dec 9.

AvrPm2 encodes an RNase-like avirulence effector which is conserved in the two different specialized forms of wheat and rye powdery mildew fungus

Coraline R Praz  1 , Salim Bourras  1 , Fansong Zeng  2   3   4 , Javier Sánchez-Martín  1 , Fabrizio Menardo  1 , Minfeng Xue  2   3   4 , Lijun Yang  2   3   4 , Stefan Roffler  1 , Rainer Böni  1 , Gerard Herren  1 , Kaitlin E McNally  1 , Roi Ben-David  5 , Francis Parlange  1 , Simone Oberhaensli  1 , Simon Flückiger  1 , Luisa K Schäfer  1 , Thomas Wicker  1 , Dazhao Yu  2   3   4 , Beat Keller  1
Affiliations

AvrPm2 encodes an RNase-like avirulence effector which is conserved in the two different specialized forms of wheat and rye powdery mildew fungus

Coraline R Praz et al. New Phytol. 2017 Feb.

Erratum in

  • Corrigendum.
    [No authors listed] [No authors listed] New Phytol. 2019 Jun;222(4):2038. doi: 10.1111/nph.15831. Epub 2019 Apr 10. New Phytol. 2019. PMID: 31066073 Free PMC article. No abstract available.

Abstract

There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race-specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3a2/f2 that is recognized by Pm3a/f was known molecularly. We performed map-based cloning and genome-wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2. The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase-like effectors, including Avra13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13. These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

Keywords: Blumeria graminis; Pm2; RNAse-like; avirulence gene; powdery mildew; wheat.

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Figures

Figure 1
Figure 1
Genetic and physical mapping of AvrPm2. (a) Linkage group 15 containing the AvrPm2 locus and the closest Kompetitive Allele‐Specific PCR (KASP) markers. Distances between markers are indicated in cM. (b) Contig 51 containing the AvrPm2 linked marker 051_LE. Dark grey boxes represent regions for which sequences are available (Wicker et al., 2013) and light grey boxes are sequence gaps. (c) Fine mapping of the AvrPm2 region. The most informative cleaved amplified polymorphic sequence (CAPS) marker and single nucleotide polymorphism (SNP) markers are indicated in black. Markers designed on the genes identified by the collinearity approach (Supporting Information Fig. S1; Notes S5) are depicted in green. Genetic distances between each marker and AvrPm2 are indicated in cM and in number of recombinants with AvrPm2. Red frames indicate misassemblies in the genome sequence. The interval containing AvrPm2 is indicated in orange. (d) Bacterial artificial chromosomes (BACs) minimal tiling path of the LTC scaffold spanning the AvrPm2 region. The markers designed on BAC end sequences and used to validate the BAC overlap are indicated with vertical grey lines and the presence of specific PCR amplifications is indicated with grey dots. The most informative markers are indicated with black and green vertical lines, and presence of specific PCR amplifications with black and green dots, respectively. The AvrPm2 interval is indicated in orange. (e) Schematic representation of the assembly of the AvrPm2 region created from four sequenced BACs. The positions of the informative markers are indicated in black. The AvrPm2 candidate gene interval is indicated in orange and other genes in green. (f) Visual representation of the mapping of genomic sequencing reads from the virulent parent JIW2 on the AvrPm2 locus assembly. Here, 50 kb up‐ and downstream of the gene BgtE‐5845 is represented. The position of BgtE‐5845 is represented with an orange vertical line and no reads map in and around BgtE‐5845, indicating a sequence deletion in the virulent parent JIW2.
Figure 2
Figure 2
Genome wide association mapping of AvrPm2 in a set of 41 Blumeria graminis (B. g.) tritici and 19 B. g. triticale isolates. (a) Genome‐wide association study (GWAS) results at the genome level. The −Log10 of the test P value of 424 219 single nucleotide polymorphisms (SNPs) after correction by λ is plotted against its physical contig position. Linkage groups are shown in different colours. Contig 51 containing the best correlated SNP is shown in green. The best correlated SNP is indicated with an arrow. (b) GWAS results at the contig level. The −Log10 of the test P value of the SNPs located on contig 51. The best correlated SNP is indicated with a vertical green line and the position of the gene BgtE‐5845 with a vertical orange line. (c) The coverage value per 1000 bp window of a 40 kb interval around the position of BgtE‐5845 (orange vertical line) is plotted. The blue lines indicate avirulent isolates and the red lines virulent isolates. In all virulent isolates, the coverage between positions 510 000 and 522 000 is < 5×, indicating a deletion of 12 kb in the virulent isolates. (d) Annotation of the region containing AvrPm2 and the 12 kb deletion in the Pm2 avirulent isolate 96224. The gene BgtE‐5845 is indicated with an orange box. Other boxes indicate transposable elements. The deleted sequence in JIW2 is indicated with a black frame.
Figure 3
Figure 3
Amino acid sequence of the protein encoded by Pm2. The coiled‐coil (CC), nucleotide binding domain (NB) and leucine rich repeat (LRR) domains are indicated on the left. Polymorphisms present in the susceptible allele are represented in red until the premature stop codon at position 423 highlighted with a red star. The amino acids represented in blue show the mutations found in the EMS mutants described by Sánchez‐Martín et al. (2016). Residues marked with ‘1’ indicate mutations found in two independent mutants. Residues marked with ‘2’ indicate mutations found in the same mutant.
Figure 4
Figure 4
Functional validation of the AvrPm2–Pm2 interaction by agroinfiltration in Nicotiana benthamiana. (a) Schematic diagram of the protein variants encoded by AvrPm2, its direct homologue in Blumeria graminis (B. g.) secalis and the variant identified in the B. g. triticale isolate CAP‐39‐A1 used for transient expression assays. ‘1’ represents the full‐length protein encoded by AvrPm2 (120 residues) and ‘2’ the variant without signal peptide. The predicted signal peptide (SP, SignalP version4.1) is indicated in green. The locations of the Y(x)xC motif and the conserved cysteine in the C‐terminal region are indicated in black and the RxFP motif in red. Blue bars indicate single nucleotide polymorphisms (SNPs) distinguishing BgtE‐5845 and the other variants. ‘3’ is the orthologue of AvrPm2 in B. g. secalis, ‘4’ and ‘5’ are two variants containing only one of the two SNPs of B. g. secalis, and ‘6’ is the variant of the B. g. triticale isolate CAP‐39‐A1. (b, c) Agroinfiltration assays in N. benthamiana demonstrate induction of the hypersensitive response (HR) upon specific recognition of AvrPm2 by Pm2. AvrPm2 without signal peptide (AvrPm2no SP) was transiently expressed together with Pm2 in a 4 : 1 Avr : R ratio. AvrPm2 and Pm2 were also co‐expressed with GUS as a control showing that AvrPm2 and Pm2 alone do not induce HR. As a positive control, AvrPm3 a2/f2 was co‐expressed with Pm3a. (d) Quantification and comparison of the HR induced upon co‐infiltration of AvrPm2 and Pm2 to that induced upon co‐infiltration of AvrPm3 a2/f2 and Pm3a, using two different Avr : R ratios (4 : 1 and 2 : 1). The fluorescence measurements are depicted in ‘integrated density’, which is the product of the area infiltrated and the mean grey value. (e, f) Agroinfiltration assays in N. benthamiana with AvrPm2 constructs encoding the protein versions with and without signal peptide co‐infiltrated with Pm2 (Avr : R ratio = 4 : 1). Stronger HR was observed with the AvrPm2 construct expressing the protein without signal peptide. (c, f) Fluorescence imaging of the HRs obtained from assays in (b) and (e) used for HR quantification. For the assays in (b)–(f) leaves of 4‐wk‐old N. benthamiana plants were infiltrated with Agrobacterium tumefaciens cultures expressing each of the constructs indicated. Results were consistent across at least three independent replicates where at least four leaves were assayed. Photographs were taken 5 d after infiltration.
Figure 5
Figure 5
Gene expression of AvrPm2 and AvrPm3 a2/f2 in the avirulent isolate 96224. (a) The mean normalized expression of AvrPm2 (red line) and AvrPm3 a2/f2 (black line) in the avirulent parental isolate 96224, 1–8 d post infection (dpi) of the susceptible wheat genotype Chinese Spring. (b) Magnification of the lower mean gene expression of AvrPm2 in isolate 96224, 1–8 dpi of Chinese Spring. Each data point is the average of three technical replicates. These results were consistent in three biological replicates as shown in Supporting Information Fig. S7. The error bars indicate the standard error of the mean (SEM).
Figure 6
Figure 6
Phylogenetic analysis of the AvrPm2 family. (a) Maximum likelihood tree of the AvrPm2 family in Blumeria graminis (B. g. ) tritici and B. g. hordei. The tree was inferred with the complete nucleotide sequences. Red branches, B. g. tritici genes; blue branches, B. g. hordei genes. The two framed boxes indicate clade 1 and clade 2. The genes highlighted in yellow are part of cluster 1 in the genetic map and those highlighted in blue are part of cluster 2. AvrPm2 (BgtE‐5845) is indicated in red, and Avr a13 (CSEP0372) in blue. The alternative names of the B. g. hordei genes CSEP0064 and CSEP0264 are indicated in parentheses. The genes tested for recognition by Pm2 by transient assays in Nicotiana benthamiana are indicated in bold. The scale bar indicates a measure of expected substitutions per site. Bootstrap values are indicated on the branches. (b) Consensus linkage group (Bourras et al., 2015) with clusters 1 and 2 indicated in yellow and blue, respectively. AvrPm2 (BgtE‐5845) is located in cluster 1. The scale bar indicates the genetic distance (cM).
Figure 7
Figure 7
Three‐dimensional protein models of AVRPM2, its closest homologue in Blumeria graminis (B. g.) hordei, and AVR a13. Three‐dimensional protein models for (a) BgtE‐5845 (AVRPM2), (b) the closest AVRPM2 homologue in B. g. hordei CSEP0066 and (c) AVR a13 (synonym: CSEP0372). (d) The known crystal structure of ribonuclease T1 from Aspergillus phoenicis, which was found to be the best template for the AVRPM2 family by the raptorX structure prediction server (http://raptorx.uchicago.edu/).
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