| Powdery mildews are major diseases on cereals such as wheat and barley in China.Wheat powdery mildew is caused by Blumeria graminis f.sp.tritici?Bgt?,which is an obligate biotrophic fungal pathogen.It is extensively distributed in wheat-growing areas of China and can disperse over long distance.Isolates can be differentiated into races to different cultivars due to frequent virulence variation.Resistant cultivar breeding and planting is one of the most economic,effective and environmental measures to control this disease.Race-specific resistance mediated by several major Pm genes has been widely used in breeding program for resistance to powdery mildews.However,new virulent isolates often occur and accumulate in populations of this pathogen due to selective pressure exerted by cultivars with vertical resistance,which give rise to break down of corresponding resistance genes.To solve this problem,two strategies can be applied.One is monitoring pathotypes of the pathogen populations and further coloning avirulence genes in order to investigate the molecular basis underlying virulence variation for more effective use of resistance genes.The other is to breed cultivars with durable resistance and accelerate this process.The aims of this study were to 1)analyze pathotype diversity of the Bgt populations in China and identify avirulence genes from isolates with divergent virulence spectrums using comparative genomics methods to uncover its variation?s?;2)characterize conidiation related genes that play key roles in asexual reproduction of Bgt by RNA-seq analyses and study their functions using gene silencing,which contribute to breeding of cultivars withdurable resistance mediated by RNA interference?RNAi?.To uncover the pathotype diversity on a nation scale,1,082 single colony isolates from 8 major wheat-growing areas were obtained in 2011 and their pathotypes on detached leaves of 22 differentials containing individual Pm genes were investigated.A total of 1,028 pathotypes were detected,of which 948 containing only one isolate.Virulence frequencies on these differential lines ranged from 0 to 97.4%.No isolate was virulent to Pm21 and less than 17.0%isolates from all the 8 populations were virulent to Pm13.In contrast,at least 56.0%isolates were compatible to Pm1a,Pm3b,Pm3c,Pm3f,Pm5a,Pm6 and Pm8.Diversity analysis revealed rich diversity of pathotypes was present within each population.the ?2 test indicated a divergent distribution of virulence frequencies of these populations to individual genotype?p<0.01?,including Pm1a,Pm2,Pm3a,Pm3b,Pm3c,Pm3d,Pm3e,Pm3f,Pm4a,Pm4b,Pm5b,PPm7,Pm8,Pml7 and Pml9,which reflect different virulence structures among these populations.Moreover,a significant positive correlation between genetic distance and geographical distance of these populations was detected based on avirulence loci?R2=0.494,p<0.001?.The population from Xinjiang province appeared to be a separate group due to its long distance from other populations.Associations among alleles at avirulence loci in pairs indicated that significant positive and negative associations between alleles at pairs of avirulence loci were detected in 26 and 31 allele pairs to Pm genes,respectively?p<0.05?.These results not only provide advice for deployment of Pm genes,but also experimental materials for cloning of avirulence genes.To clone avirulenc genes,we sequenced the genome of Bgt isolate 21-2 using the next?Illumina?and third generation?PacBio?sequencing platforms.De novo assembly was performed and gene prediction and annotation were accomplished after remove of repeat sequences.Ninety-nine isolates with divergent virulence patterns were selected and their genomes were re-sequenced.Genetic variations among these genomes including SNP?single nucleotide polymorphism?,InDel?insertion and deletion?and PAV?presence/absence variation?were investigated by mapping to the reference genome.To clone AvrPm2,an association study between their effector genotypes and the phynotypes to Pm2 were conducted.Afterwards,PCR validation,sequence analysis,and expression pattern analysis of the candidate gene was carried out.In result,a 132.4Mb genome could be assembled with 2,654 scaffolds and N50 length of 80.9kb.The assembly quality of the genome of isolate 21-2 was significantly improved compared to that of isolate 96224.After masking the repeat sequences?74.7%?and non-coding RNA?0.06%?,we found 6,770 protein coding genes from the rest of the genome sequence,of which 6,743 genes were annotated.Identification of effector genes predicted 644 effectors in isolate 21-2,including 544 candidate secreted effector proteins?CSEPs?and 192 candidate effector proteins?CEPs?.Analysis of re-sequencing data identified a large number of SNPs,InDels and PAVs.We found PAVs with more than 100bp and more than 500bp in length in 58 and 30 effector genes among these isolates,respectively.A Fisher's exact test based on both PAVs and phenotypes of 100 isolates to Pm2 revealed that PAVs of 4 genes were significantly associated with AvrPm2 phenotypes?p<0.01?.One gene,Bgt21-2005279,had the smallest p value.A manual inspection confirmed that the PAVs of this gene in 97 isolates coincided exactly with the variation of AvrPm2 phenotypes of these isolates,strongly suggeting that this gene is the first candidate of AvrPm2.A 12kb deletion in the region where the gene located resulted in the virulence gain of avirulent isolates to Pm2.Some transposons belonging to the long terminal repeat?LTR?family were found in the deletion region around this gene.Two isolates without this gene were avirulent to Pm2,yet one isolate with this gene was virulent to Pm2,which suggested that other variation?s?of this gene or other genes might be implicated in avirulence or virulence to Pm2.Sequence analysis indicated that this gene encoded an RNase-like effector with 119 amino acids in length.A predicted signal peptide was present in the N-terminal region of this protein,followed by a conserved Y?x?xC motif and a RxFC motif.Transcriptome analyses revealed that this gene was highly expressed in haustoria at 2dpi?days post inoculation?.Our results suggest that RNase-like effectors play important roles in virulence variation of cereal powdery mildews.To uncover molecular mechanisms underlying conidiation of cereal powdery mildews,we sequenced the transcriptome from the epiphytic tissues of isolate 21-2 at 3?vegetative hyphae growth?dpi,4?foot cells initiation?,and 5?conidiophores formation?dpi.RNA-seq analyses showed that 556 and 404?a total of 685?genes were differentially expressed at 4dpi and 5dpi during conidiation in comparison to their expression levels at 3 dpi,respectively.We found that several primary metabolism related genes that were involved in the conversion from various sugars to glucose,glycolysis,the trcarboxylic acid cycle?TAC?,the electron transport chain?ETC?,and unsaturated fatty acid oxidation were activated,which suggest that more energy is required during Bgt conidiation.Meanwhile,glucose was converted into glycogen,which was accumulated in developing conidiophores,indicating that it could be used as the primary energy storage molecules in the next infection cycle.Cluster analysis of 91 regulatory genes based on their expression patterns revealed that calcium?Ca2+?,H2O2,and phosphoinositide?PIP?signaling pathways were implicated in Bgt conidiation.Treatment of EGTA,a Ca2+ chelator,and trifluoperazine dihydrochloride?TFP?,a calmodulin?CaM?antagonist,significantly suppressed the accumulation of H2O2,impaired foot cell and conidiophore formation and reduced conidia production of Bgt.These results suggest that Ca2+ and H2O2 mediated signaling play vital roles in conidiation and they are crosslinked with each other.In addition to some conidiation-related orthologs known in other fungi,we found some novel B.gramminis-specific genes,reflecting that a unique molecular basis underlying asexual sporulation of B.graminis.RNA-seq analysis during Bgt conidiation identified a protein kinase coding gene that might act in Ca2+ or H2O2 mediated signaling.Sequence analysis of this gene showed that it encoded a KSP1 serine/threonine protein kinase?STPK2?with a predicted nuclear localization signal.To further determine its role during conidiation,we have the endogenous transcript of this gene silenced using virus-induced gene silencing?VIGS?.Quantitative PCR analysis demonstrated that the expression level of STPK2 was reduced significantly at 5dpi?p<0.01?.Histological investigation showed that silencing of this gene caused significant reduction of the formation rate of foot cell and conidiophore and conidia production?p<0.01?,which resulted in obviously reduced disease symptoms.These results suggest that STPK2 is essential for Bgt conidiogenesis and can be used as a candidate target gene in breeding for durable resistance. |
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