Study On The Pathogenic Mechanism Of Soybean Phyton Effector PSR

Soybean(Glycine max)is crucial food and economic crop in our country.Throughout its growth and development,soybean faces infection from various pathogens,resulting in disease occurrence and substantial losses.Among these diseases,root rot caused by Phytophthora sojae stands out as one of the most severe soil-borne ailments worldwide,inflicting considerable damage on soybean production annually.Research has revealed that Phytophthora sojae promotes infection by secreting toxic effectors to the host plant while interfering with diverse plant signaling pathways.However,there are currently no effective treatments or disease-resistant varieties available for preventing and controlling this devastating disease.Therefore,studying the pathogenic mechanism of these effectors holds immense significance in safeguarding soybean production and fostering the sustainable growth of this industry.An effector Phytophthora suppressor of RNA silencing 1(PSR1)that can inhibit plant RNA silencing was previously identified in the laboratory.Further studies found that PSR1 interacted with plant pre-mRNA splicing factor 16(PRP16),also known as PSR1-Interacting Protein 1(PINP1).PSR1 overexpression and PINP1 silencing plants showed a decrease in sRNA abundance.Moreover,expression of PSR1 in Arabidopsis thaliana and Nicotiana benthamiana promoted Phytophthora capsici infection,and increased Phytophthora sojae colonization in soybean.However,the molecular mechanisms by which PSR1 regulates sRNA production and plant disease resistance remain unclear.In this study,we firstly knocked out PSR1 from the genome of P.sojae by CRISPR/Cas9 technology,and identified three PSR1-knockout homozygous transformants by PCR.Plate growth assays showed that the PSR1-knockout transformants did not affect its growth and development.The results of soybean hypocotyl inoculation showed that the pathogenicity of PSR1-knockout P.sojae transformants was significantly lower than that of wild type P.sojae,which confirmed that PSR1 is very important in the virulence of P.sojae during the infection of the host.We next analyzed the PINP1 homologous protein sequences in animals,plants and oomycetes,and found that PINP1 and PINP1 homologous proteins have low similarity at the N-terminus,while the C-terminus is very conservative.Meanwhile,all PINP1 homologous proteins contain a DEAH-box RNA helicase domain.Yeast two-hybridization and BiFC assays showed that PSR1 interacts with all detected PINP1 homologous proteins,and the three conserved domains at the C-terminus of PINP1,DEAH,HA2 and DUF1605,were the key domains for its interaction with PSR1.Because the sequences of PINP1 and its homologous proteins are relatively conservative,we performed yeast complementation assay,and the results showed that PINP1 could complement the growth defect phenotype of prp16-deficient yeast strains.These results suggested that plant PINP1 and yeast PRP16 may be functionally conservative.The sequence of PINP1 was analyzed by bioinformatics and found to contain a conserved DEAH-box RNA helicase domain.In vitro RNA helicase assay confirmed that PINP1 is a class of RNA helicase that mainly provides energy by hydrolyzing ATP.The ATPase activity experiment showed that PINP1 had ATP hydrolysis activity and the ATPase activity of PINP1 mutant was affected.The enzyme activity of PINP1 and its mutant required binding to total ArabidopsisRNA.RNA EMSA revealed that PINP1 bound to general 80 nt sRNA(ssRNA and dsRNA).Previous studies have shown that sRNA accumulation is significantly reduced in PINP1 silenced and PSR1 overexpressed plants.In order to reveal the molecular mechanism of PSR1 and PINP1 regulating sRNA production,we selected 26 sRNA pathway genes and conducted protein interaction analysis.Yeast two-hybrid assays showed that neither PINP1 nor PSR1 could interact with key components of sRNA pathway.Through RNA EMSA and RNA IP assay,we confirmed that PINP1 bound to pri-miRNA to regulate miRNA production in plants.Interestingly,we found that in vitro helicase assay and RNA EMSA showed that PSR1 inhibited PINP1’s helicase activity and RNA binding activity,and also confirmed that PSR1 inhibited PINP1’s ability to bind to pri-miRNA.RNA pull-down assay further confirmed that PSR1 inhibited PINP1’s binding to pri-miRNA.These results suggested that PSR1 interferes with PINP1’s ability to bind to pri-miRNA by interacting with PINP1,thereby regulating sRNA production in plants.PINP1 gene predicted to encode pre-mRNA splicing factor PRP16.In order to investigate the role of PINP1 on pre-mRNA splicing and the effect of PSR1 on PINP1 splicing ability.We performed RNA-seq analysis on plants with PSR1 overexpressed and PINP1 silenced.The results showed that a large number of genes had alternative splicing(AS)events in PINP1 silenced and PSR1 overexpressed plants,among which intron retention(IR)was the most common type of AS.RT-PCR and qRT-PCR assays also verified the existence of AS genes and events in PINP1 silenced and PSR1 overexpressed plants.These results suggested that PSR1 may regulate Arabidopsis AS by interacting with PINP1.In order to determine whether splicing isoforms of the intron retention can be translated into abnormal proteins.We further verified by Western bolting that the IR splicing forms of DCL2 and DCL3 produced truncated proteins in PINP1 silenced and PSR1 overexpressed plants.We identified five sRNAs and JA signaling pathway-related genes(AGO4,CPL4,AOC2,OPCL1 and MES10)that had intron retention and were involved in plant defense responses.We clarified that the truncated proteins of IR genes could enhance plant infection with Phytophthora.In summary,these results revealed that the Phytophthora sojae effector PSR1 inhibited the ability of PINP1 to bind to pri-miRNA and pre-mRNA splicing to regulate sRNA production and plant defense responses by interacting with PINP1.