| Wheat is one of the most important crops in the world, and powdery mildew is a main damaging disease in wheat. The quick pathogen variants result in the loss and unendurable of race-specific resistance of wheat. Genetically, more than40resistance genes (Pms) have been identified in wheat in China, but just the Pm3family genes were cloned in the world. Cloning and congregating most or all of these genes in a cultivar will be time-consuming and very arduous. In previous study, some host genes were found to be required for successful infection of biotrophic pathogens in plants, known as susceptibility genes or susceptibility factors. Loss-of-function mutations in these genes lead to increased resistance to a particular pathogen or multiple types of pathogens. The identification of susceptibility genes will open a new potential way for plant resistance improvement. However, no genes conferring susceptibility to powdery mildew in wheat were reported. Hence, genechip hybridization experiments were performed to examine genes differentially expressed in resistant and susceptible wheat cultivars with powdery mildew inoculation, and further transient single-cell expression technique was used and identified three genes involved in susceptibility. The results are as follows:1. The histology and cytology results showed that after powdery mildew inoculation, there is very high haustorium formation frequency in Yumai13with susceptible symptom. However, the cultivars Hongyou and Chiyacao were resistant to powdery mildew with papillae formation and hypersensitive response occurrence.2. Total RNA was extracted from different wheat cultivars with powdery mildew inoculation and used in genechip hybridization. Analyzing genechip data, eleven candidate genes were screened which were just expressed in the susceptible wheat both after inoculation and pathogen infection, but nearly no expression in resistant cultivars. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses were carried out to confirm the microarray hybridization results. The cDNAs corresponding to the transcripts were cloned and used to construct RNAi vectors suitable for suppression of the corresponding endogenous genes using biolistic transient single-cell transformation of epidermal cells. Our study demonstrated that expression of three RNAi constructs could increase resistance, suppressing of other eight genes had no significant effect on the relative penetration efficiency, even had opposite effect.3. Our research identified two susceptibility genes represented long non-coding RNAs corresponding to EST accession no. BQ168479and CA648596, dubbed TaSl and TaS2(Triticum aestivum susceptibility, TaS). Suppressing of the two genes could increase resistance by22%and15%respectively. Full-length cDNAs were obtained and sequenced by RACE experiment. Significant changes were observed in different organs in the susceptible wheat by qRT-PCR analyses, and they could be markedly accumulated and played roles during powdery mildew primary infection. Blast analysis showed no homology genes were checked out in other plants.4. Suppressing of the corresponding endogenous gene and stable over-expressing of TaS3using biolistic transient expression system, the resistance was influenced by19%and14%respectively, by the way of penetration efficiency compared to control. The full-length cDNA of TaS3was obtained and predicted to encode a protein of381amino acids of the putative ULP1peptidase, which is a SUMO specific protease. Subcellular localization studies revealed that the protein encoded by this gene was in the nucleus. qRT-PCR analyses revealed that TaS3transcript is significantly induced at12hpi, that means ULP1protease was up-regulated. TaS3may regulate SUMO function positively related to pathogen attack. |
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