Screening Of Important Genes Related To Urediniospore Germination And Their Expressional Analysis In The Early Infection Stage Of Puccinia Triticina

Wheat leaf rust,caused by Puccinia triticina Eriks（PT）,is an destructive disease worldwide.The most economical and effective way for controlling leaf rust disease is to cultivate resistant wheat varieties.However,virulent variation of Puccinia triticina often leads to the loss of resistance of wheat varieties,resulting in the repeated outbreak and epidemic of leaf rust.Therefore,screening the genes related to pathogenicity of Puccinia triticina is of great significance for analyzing pathogenic mechanism of leaf rust and developing new wheat varieties with broad-spectrum resistance to leaf rust.Urediniospore germination of Puccinia triticina is an important development stage in the early stage of infection.Some important genes induced by germination may play an important role in the successful infection of Puccinia triticina and its early interaction with wheat.In this study,PacBio single molecule real-time sequencing technology was used to sequence the full-length transcriptome of spores and germ tubes from leaf rust urediniospores germinating for 0 h,2 h,4 h,8 h,12 h and 24 h.At the same time,high-throughput second-generation sequencing technology was used to detect expression levels of related genes at different time points.Differential expression analysis and biological information analysis of full-length transcripts were integrated,the important genes in the germination process of urediniospores were screened,and their expression patterns in different virulence isolates and the early stage of affinity/incompatibility infection were systematically analyzed.The main results are as follows:1.Total RNA was extracted from mixed samples of the spore and germ tube urediniospores germinating at 0 h,2 h,4 h,8 h,12 h and 24 h.And then,the full-length transcriptome was sequenced using PacBio single molecule real-time sequencing technology.The results showed that a total of 20853 high-quality transcripts were obtained,with an average length of 2170.51 nt.12035 transcripts containin intact CDS,and 623 alternative splices,59 long noncoding RNAs（lncRNA）and 7552 SSRs were identified.NR,Swiss-prot,GO,COG,KOG,Pfam,KEGG and eggNOG were used to annotate the transcripts,and 97%of the transcripts were annotated.2.Using Illumina sequencing technology,we sequenced the transcriptome of samples at six time points of urediniospores germination,analyzed the difference in the expression at each time point,and screened the genes with more than twice the expression difference as the significantly differentially expressed genes.Compared with resting urediniospores（0 h）,5121,2329,2424,3199 and 3699 genes were differentially expressed at 2 h,4 h,8 h,12 h and 24 h,respectively.Based on the bioinformatics analysis of the full-length transcripts of the three generations of different genes,four candidate genes in the germination process were selected.PTTG₁050,PTTG 4765 and PTTG 1279 are up-regulated differentially expressed gene during germination,PTTG 1823 is a down regulated differentially expressed gene during germination.The domain of PTTG 1050 is annotated as syntaxin-6_N superfamily and SNARE superfamily,suggesting that it may be involved in SNARE（Soluble N-ethylmaleimide-sensitive factor attachment protein receptors）mediated vesicle transport,and related to the development process of leaf rust;The domain of PTTG 4765 is annotated as 3a01205 superfamily（Pleiotropic drug resistance family protein）,which may play an important role in helping pathogens resist adverse environment;The domain of PTTG₁823 is annotated as PKC_Like（protein kinase C-like）superfamily which participates in MAPK signaling pathway,and may be related to rust germination/infection;PTTG₁279,a secreted protein,was identified in this study may play a role in pathogen invasion and resisting host defense response.3.Six samples of wheat leaf rust fungus were collected from Jiaozuo,Luohe,Xuchang,Shangqiu and Zhengzhou,Henan Province.They were isolated,purified and propagated in the laboratory,and six single spore lines of wheat leaf rust fugus were obtained.Virulence identification was carried out on 19 wheat varieties with different genotypes.Combined with the phenotypic symptoms of leaf rust strains infecting different wheat varieties for 12 d and the accumulation of rust biomass in wheat infected leaves,three strains with different virulence were identified,which were named after PT-1,PT-2 and PT-3 respectively.Pt-1 had the widest toxicity spectrum and the strongest pathogenicity,PT-2 followed,PT-3 had the narrowest toxicity spectrum and the weakest pathogenicity.4.The highly virulent strain Pt-HnZU18-3 of leaf rust was inoculated into 6 different wheat varieties to identify the expression patterns of four candidate genes.PTTG 1050,PTTG 4765 and PTTG 1823 were significantly up-regulated in susceptible varieties at different time points after inoculation;PTTG₁279 was significantly up-regulated in resistant/susceptible varieties.The expression patterns of candidate genes in the early infection of leaf rust strains with different virulence were analyzed by using three isolated single spores of leaf rust strains to form incompatible and compatible combinations with Yunmai 34 and Chinese spring wheat varieties,respectively.PTTG 1050 and PTTG 4765 were significantly up-regulated in the compatible combination of virulent strain PT-1.In the incompatible combination,the two candidate genes were also induced up-regulated,too.However,in the incompatible combination,the peak expression was 12h,while in the compatible combination,the peak expression was 24h,and the overall expression of candidate genes in the compatible combination was higher than that in the incompatible combination.It is speculated that PTTG₁050,PTTG₄765 may be related to the strong virulence of leaf rust and play an important role in the growth and development of rust.PTTG 1823 was significantly up-regulated in the compatible combination of PT-1 and PT-2,but down regulated in the incompatible combination of PT-1 and PT-2,indicating that it may be closely related to its smooth expansion in the susceptible host,while its expression was inhibited in the resistant host,indicating that it is also related to the toxicity of leaf rust.PTTG 1279 was significantly induced during the infection of moderately virulent strain PT-2.In the compatible combination,it reached the peak at 8 h and continued to be highly expressed at the subsequent time points.In the incompatible combination,it reached the peak at 12 h and then decreased rapidly;PTTG₁823 and PTTG 1279 showed significantly different expression patterns in the early infection of different virulent strains,indicating that it may have its own unique pathway or mechanism in different virulent strains.In this study,four candidate genes that may play an important role in the early stage of infection were identified by transcriptome sequencing analysis of urediniospores of Puccinia triticina and qRT-PCR analysis of related genes within 24 hours of infection,which provided an important basis for understanding the early interaction mechanism of leaf rust infection.