Diversity Of Small RNAs And Their Potential Roles In The Regulation Of Gene Expression In Phytophthora Parasitica

Oomycetes represent a large group of fungus-like filamentous eukaryotic microorganisms that are phylogenetically separated from fungi,plants and animals.The representative genus,Phytophthora spp.,includes some of the most destructive plant pathogens causing huge economic losses worldwide.Owing to the unique phylogenetic process,oomycetes differ greatly from fungi in physiological and biochemical characteristics.This is responsible the ineffectiveness of most fungicides against oomycetes.Furthermore,the use of resistant cultivars is an important strategy in plant disease control.However,Phytophthora pathogens evolved quickly and led to loss of disease resistance of many resistant cultivars.The avirulence genes are frequently lost or mutated to avoid being recognized by corresponding resistance genes.However,the avirulence genes in some virulent Phytophthora strains showed no any changes in sequence but in expression,suggesting that epigenetic regulations may play important roles in virulence variations in Phytopthora.Therefore,we chose P.parasitica as a model species to examine diversity of small RNAs in Phytophthora and to explore their potential roles in the regulation of gene expression in P.parasitica.Meanwhile,we took advantage of the P.parasitica-Nicotiana benthamiana model interaction system and transcriptome approach to understand compatible plant-Phytophthora interaction.The main results are as follows:1.We used high throughput sequencing technologies to examine diversity of small RNAs in Phytophthora and found:a)Besides the infection-induced tRNA-derived small RNAs(sRNAs),there are two distinct types of sRNAs in P.parasitica,which are 25-26 nt and 21 nt respectively.They can be mapped to genome in cluster without significant strand-biases and have strongly base preference at the 5' terminal.b)There are more than 7000 endogenous gene loci generate 25-26 nt sRNAs.RNA-seq results the reliability of which is confirmed by quantitative PCR,show that most of these 25-26 nt sRNA-associated genes are not expressed at both of vegetative growth and infection stages.The sRNAs can be detected in the exon,intron,upstream and downstream of gene regions in similar levels,indicating that the silencing may occur in transcriptional level,but not post-transcriptional level,c)The 25-26 nt sRNA-associated genes are typically P.parasitica species-specific,with no homologies to the sister species P.infestans.They include approximately 40%RXLR effector genes,50%Crinkler effector genes and some elicitor genes.These results indicate that 25-26 nt sRNAs may play important roles in the silencing of new genes and virulence variations in Phytopthora.d)The 25-26 nt sRNA generally arise from gene-sparse and repeat-rich regions.And the sRNA generation and gene silencing status can spread from repeat regions to adjacent regions about hundreds to thousands of base pairs.Most of the 25-26 nt sRNA-associated genes lie in or are very close to repeat regions.e)The 21 nt sRNAs typically are mapped to the exon sequences of high-expression genes,suggesting that the biogenesis of the 21 nt sRNAs may depend on gene expression and the amounts are insufficient to completely silence genes,f)Analyses of the published P.infestans sRNA and mRNA sequencing data consistently show that the 25-26 nt sRNAs,but not the 21 nt sRNAs,are associated with efficient silencing of homologus endogenous genes in P.infestans,suggested that the mechanisms are conserved in Phytophthora.2.To understand the Phytophthora-plant interaction mechanisms,we performed RNA sequencing of N.benthamiana leaf samples at 0,3,6,12,24,48 hours after inoculation of P.parastica zoospores,and found:a)Transcriptome results show that the gene expression levels at early and late infection stages are significantly different.The genes up-regulated at early infection stage typically are down-regulated at the late stages.Therefore,we identified 231 genes encoding secreted proteins and specially up-regulated at early infection stage,including large number of RXLR effector genes,NPP-like elictor genes,hydrolase and protein inhibitor genes.We also found that most of them belong to multigene families,including the gene families encoded carbonic anhydrases,berberine-like and SCP-like proteins and some families with unknown function.We analysed a family systematically and found that gene duplication and domain rearrangement of this family members occurred frequently in Phytophthora and it could be divided into three sub-families based on the domain architecture.c)A large number of genes related to protein translation,nucleosome assembly,RNA methylation are up-regulated at early infection stage.And the genes related to tricarboxylic acid cycle and ATP generation are up-regulated at medium and late stages.However,it is not expected that the genes related to vesicle-mediated transport,microtubule-based movement,protein ubiquitination and signal transduction are down-regulated at early infection stage.3.To understand the molecular mechanisms of resistance to Phytophthora,we used RNA-seq data to perform transcript assembly and differential gene identification,and found a)A large number of defense response genes including PAMP-indeced genes and genes related to reactive oxygen species and programmed cell death,are up-regulated at infection stages.The genes related to the biogenesis and signal transduction of salicylic acid,jasmonic acid,ethylene are also up-regulated at infection stages.Most of the hormone-related genes and the flg22/elf18 induced genes are induced in infection and show two expression peaks in 3hpi and 24hpi.Thus,we identified 3766 up-regulated genes with similar expression pattern which were important candidate defense genes.Among of them,753 genes are up-regulated more than 10 fold at 3hpi.c)Meanwhile,we systematically identify a large number of protein kinases and transcription factors most of which are induced in infection and show two expression peaks in 3hpi and 24hpi.A series of receptor-like protein kinase and 4 receptor-like protein genes which are induced in infection and show two expression peaks in 3hpi and 24hpi are also identified.They may play important roles in PAMP/elicitor recognition and signal transduction.d)The expressions of a large number of genes related to protein secretion,protein modification and catabolic process are induced at infection stages.And the expressions of genes related to photosynthesis,proton transport and the biogenesis of protein,fatty acid and carbohydrate are down-regulated.