The Epigenetic Mechanism Analysis Of Avirulence Gene Silencing In Phytophthora Sojae

Phytophthora sojae belongs to oomycete.Phytophthora root rot(PRR)caused by P.sojae is one of the devastating diseases threatening soybean production security.This disease is characterized of being easy to break out and its pathogen is easy to mutate.Researching the pathogenic and mutation mechanisms of P.sojae is of great significance to the prevention and control of the disease.The interaction between P.sojae avirulence gene(Avr)and soybean resistance gene(Rps)is in line with the gene-for-gene hypothesis.The mutation of avirulence genes directly determines the succession of different P.sojae pathogenic populations in the field.Therefore,to analyze the mechanism of the avirulence gene mutation of Phytophthora and clarify its evolution is an important basis for the rational and effective use of crop disease resistance genes.Previous studies have shown that epigenetic chromatin modifications including histone modification and DNA methylation are important factors that regulate the expression of important genes of plant pathogens,affecting the growth and development,metabolism,and pathogenicity of pathogens,but there are few reports related to the pathogenic differentiation of Plant Phytophthora regulated by epigenetic regulation.This article focused on the mutational phenotypes of P.sojae avirulence(Avr)gene transcription of Avr1 b and Avr3 a,comprehensively using Ch IP-seq,CRISPR/Cas9 gene editing,pathotype detection,and other methods to explore the variation mechanism.We found that P.sojae exploits epigenetic mechanisms to regulate the silencing of avirulence genes,which in turn helps to overcome host resistance mediated by resistance genes.This study reveals a new mechanism of Phytophthora overcoming host resistance,which has important reference value for the in-depth study of the natural variation of other pathogens and provides new ideas for the scientific control of plant Phytophthora diseases.Histone methylation H3K27me3 involved in the silencing of avirulence gene Avr1 b in Phytophthora sojae.The relentless adaptability of pathogen populations is a major obstacle to effective disease control measures.Increasing evidence suggests that gene transcriptional polymorphisms are a strategy deployed by pathogens to evade host immunity.However,the underlying mechanisms of transcriptional plasticity remain largely elusive.Here we found that the soybean root rot pathogen P.sojae evades the soybean Resistance gene Rps1 b through transcriptional polymorphisms in the effector gene Avr1 b that occur in the absence of any sequence variation.Elevated levels of histone H3 Lysine27tri-methylation(H3K27me3)were observed at the Avr1 b locus in a naturally occurring Avr1b-silenced strain P6497 but not in an Avr1b-expressing strain sc10,suggesting a correlation between this epigenetic modification and silencing of Avr1 b.To genetically test this hypothesis,we edited the gene,Ps Su(z)12,encoding a core subunit of the H3K27me3 methyltransferase complex by using CRISPR/Cas9,and obtained three deletion mutants.H3K27me3 depletion within the Avr1 b genomic region correlated with impaired Avr1 b gene silencing in these mutants.Importantly,these mutants lost the ability to evade immune recognition by soybeans carrying Rps1 b.These data support a model in which pathogen effector transcriptional polymorphisms are associated with changes in chromatin epigenetic marks,highlighting epigenetic variation as a mechanism of pathogen adaptive plasticity.Histone methylation H3K27me3 involved in the simultaneous silencing of avirulence gene Avr1 b and Avr3 a in Phytophthora sojae.Based on the first part of this thesis,here we further verified the causal relationship between H3K27me3 modification and avirulence genes silencing in a different strain ACR10.The expressions of Avr gene Avr1 b and Avr3 a were both silenced in the P.sojae strain ACR10,and H3K27me3 was highly accumulated on these loci,meanwhile,ACR10 evaded immune recognition by soybeans carrying cognate Rps1 b or Rps3 a gene.Deletion of Ps Su(z)12 sequence fragments in ACR10 led to the depletion of H3K27me3 modification on Avr1 b and Avr3 a loci,released the silencing of gene expression.Correspondingly,these mutants lost the ability to evade the immunity from resistant host plants carrying Rps1 b or Rps3 a.We further did sequence complementation for pssu(z)12 deletion mutants,the expressions of Avr1 b in those six complements without recombined tags were all highly significantly downregulated or even re-silenced,but only three of them significantly restored the Avr3 a silencing,indicating that the silencing mechanisms between Avr1 b and Avr3 a were not completely overlapped.Especially for Avr3 a,its gene silencing may be regulated by unknown mechanisms.This study further revealed that the modification status of H3K27me3 at multiple avirulence gene loci in P.sojae strains are closely related to their gene expression levels,and the transcriptional regulation of different loci has common mechanisms but is also likely to have natural differences.Oscillation of Avr gene expression in single-zoospore isolates of Phytophthora sojae.The two previous parts of this thesis found that the H3K27me3 involved in maintaining the silencing of P.sojae avirulence gene Avr1 b and Avr3 a,revealed a new mechanism of Phytophthora mutation at the level of epigenetic regulation.The epigenetic phenomenon is usually affected by the environment and is reversible.Previous studies have shown that single zoospore isolates can switch in virulence phenotype towards resistance host in a reversible way,but the transcript levels of Avr genes were unknown.In this study,single zoospore isolation was conducted for six P.sojae races and about 16-19 single zoospore isolates were obtained respectively.Then the transcript level changes of four avirulence genes Avr1 a,Avr1b,Avr1 c and Avr3 a in these zoospore isolates were investigated.We found that the expression status of four avirulence genes in singlezoospore isolates from strain ACR10、ACR16 and P6497 maintained stable,while in the single-zoospore isolates from strain ACR6 、 ACR9 and ACR20,the avirulence gene expression oscillated in varying degrees.It counted twice for gene Avr1 b to change from transcription to silencing in the single-zoospore isolates from ACR9 and ACR20 respectively,and counted once for Avr3 a to change from silencing to transcription in the single-zoospore isolates from ACR6.However,no similar change was observed for Avr1 a and Avr1 c,indicating it may be strain-specific and gene-specific for the expression oscillation of avirulence genes.The above studies revealed that there is obvious gene expression oscillation of P.sojae avirulence genes in the asexual offspring.It is difficult to occur sequence variations of the avirulence gene epitopic locus or trans-acting elements in a short time,so this phenomenon is probably to come up at the level of epigenetic regulation of the pathogen,suggesting that Phytophthora is likely to undergo pathogenic natural mutations in a short period time only through the process of clonal propagation.In summary,the three parts of this thesis show that P.sojae avirulence genes could cause self-silencing through a novel epigenetic regulatory mechanism mediated by histone H3K27me3,thereby evading the identification of resistant hosts.And the expression of silenced avirulence genes could be restored in the offspring,indicating the high complexity and flexibility of the epigenetic regulatory mechanism,which is likely to be a low cost of field suitability for pathogen mutation.This discovery is important for guiding the sustainable prevention and control of crop diseases in the future.