Functions Of Phytophthora Sojae RXLR Effector Avh94 During Infections And Mechanisms Underlying MiRNA Mediated Soybean Resistance To Phytophthora Sojae

Phytophthora sojae Kaufmann and Gerdemann is a member of the class oomycete,leading an annual economic loss about 1 to 2 billion dollars worldwide by causing stem and root rot on soybean,which is one of the most destructive diseases to soybean industry.Due to a lack of understanding of its pathogenesis,which limits the development of disease control and management.Investigating molecular mechanisms underlying Phytophthora sojae pathogenesis is our first priority.It is known that during infection on soybean,Phytophthora sojae secrets a large number of effectors to attack plant immune system,among which the most abundant group of effectors is called RXLR effectors.This group of effectors carries a signal peptide following by a conserved RXLR(Arg-any-Lys-Arg)motif.RXLR effectors can enter plant cell by binding PI3P on cell membrane through the RXLR motif.Bioinformatics analysis found that the genome of the P.sojae encodes about 400 candidate effectors carrying the RXLR-dEER motif.In our previous study,RXLR effectors can disarm plant immunity system by accurate transcriptional profiling and functioning synergistically.However,the mechanism underlying how these effectors interact with host proteins and interfere with plant immune system is largely unknown.In this study,we further elaborated the function of a RXLR effector Avh94 which is upregulated during the early stage of infection.Avh94 is an effector that widely exist in Phytophthora species,and it can directly interact with a repressor of JA signaling called JAZ protein.Avh94 promotes infection by targeting JAZ protein and inhibiting JA signaling during infection.In addition,it has been reported that miRNAs in the soybean are the master regulators of NB-LRR genes.In this study,we investigate the function of NB-LRR gene regulator miRNAs and found that miR1510a1b-3p is one of the major regulators of soybean NB-LRR genes,and it involves in soybean resistance to P.sojae.Functional analysis of RXLR effector Avh94.In this study,we found two copies of Avh94 genes in the genome of P.sojae.During infection,one copy named Avh94a is highly induced during early infection stage and reaches the maximum expression at 3 h post inoculation,however,expression of the other copy Avh94b remains a very low level throughout the life cycle.Avh94 has no polymorphism among the four physiological races of P.sojae;and has homologous protein in many Phytophthora species,such as Phytophthora melonis,Phytophthora kernoviae,Phytophthora taxon totara,Phytophthora capsici,Phytophthora ramorum,Phytophthora infestans,Phytophthora parasitica and P.cinnamomi.Homologs of Avh94 from different species all preserve the ability to suppress cell death induced by XEG1,a PAMP from P.sojae.Expressing of Avh94 in soybean hairy roots promotes the Phytophthora infection,indicating that Avh94 is a virulence effector in infection.But the CRISPR/Cas9 knock out mutant of Avh94 still maintain the same infection ability as the wild-type control.Taken together,it is demonstrated that Avh94 is a virulence factor in infection but the virulence function is not essential for infection,this probably due to the functional redundancy genes in P.sojae genome.Molecular target identification of Avh94 and mechanism underlying interaction between Avh94 and its interacting protein.To better understand the virulence mechanism of PsAvh94,we performed a yeast two-hybrid(Y2H)screening using P.sojae-infected soybean hypocotyl cDNA library and investigated proteins potentially associating with PsAvh94's virulence functions in host cells.One gene repeatedly identified in three independent screens encodes a protein called JAZ proteins,which is repressor protein in JA signaling.It suppresses JA response genes by interacting with transcription factors that related to the expression of these JA response genes.Upon the conception of JA related molecules,JAZ protein will be recruited by COI1 and degraded through 26s proteasome.Without the transcription inhibition by JAZ protein,JA response genes will be upregulated.We validated the interaction between Avh94 and GmJAZ1 by yeast two-hybrid,in vitro pull-down and bimolecular fluorescence complementation assay.With the truncation mutant of Avh94,we found that N terminal region of Avh94 is both required and essential for interaction with GmJAZ1,and we identify that another three effectors that can interact with GmJAZ1 in yeast,which is Avr1a,Avr1c and Avh23.Deletion of Jas domain in GmJAZ1 will disrupt its interaction with Avh94,and Jas domain alone is sufficient for interacting with Avh94.We demonstrated that Avh94 can interact with 11 JAZ protein in Arabidopsis and Arabidopsis mutant expressing Avh94 showed higher susceptibility to Phytophthora.JA signaling marker gene AtJAZ9 is significantly suppressed in Avh94 expression transgenic Arabidopsis upon the infection of Pseudomonas syringae.And soybean JA response gene Gm VSPa and GmPR3 are also suppressed in soybean hairy root which expressed Avh94.In Phytophthora infection,JA signaling is induced upon the infection of P.sojae in the early stage of infection,and overexpression GmJAZ1 protein in Nicotiana benthemiana leaves will make it more susceptible to Phytophthora,it means that suppression of JA signaling is beneficial to infection.Overall,these results demonstrate that Avh94 represses JA signaling during the early stage of infection,and it's still not clear whether this virulent function is related to JAZ protein,miRNA regulate resistance genes in soybean and mediate soybean resistance to Phytophrora sojae.miRNAs of Soybean are predicted to target hundreds of defense-related genes.miRl 507c-3p,miR1510a/b-3p,miR2109-5p,miR2118a/b-3p can directed target more than 100 NB-LRR genes in soybean.In this study,we utilized short tandem target mimic technology to silence miRNA in soybean and overexpress miRNAs in soybean hairy roots.We perform sRNA-sequencing and RNA-sequencing on these miRNAs modified soybean hairy roots.We found that miR1507c-3p miR1510a/b-3p and miR2109-5p are three major miRNAs that regulate soybean NB-LRR genes in soybean.and more than 35 NB-LRR genes are directly targeted by each miRNA.During phytophthora infection,the abundance of miR1510a/b-3p reduced significantly.Silencing miR1510a/b-3p in soybean hairy roots will promote the infection of Phytophthora.However,the transcript abundance of NB-LRR genes does not show a significant change in miR1510a/b-3p silenced hairy root,but we notice a very significant abundance change of the secondary siRNA generated from NB-LRR genes.Taken together,miR1510a/b-3p is an important regulator of soybean resistance to Phytophthora sojae,and this regulation probably related to the secondary siRNAs that generated from NB-LRR genes.Plant cyclophilin can interact with several phytophthora effectors.Cyclophilin belongs to a class of proteins that are ubiquitous in organisms and has multiple biological activities,including the activity of prolyl-peptidyl isomerase(PPIase).This group of proteins can interact with substrate and lead to a conformation change of substrate through PPlase activity.It has been reported that pathogens effectors can be activated by a cyclophilin protein in plant.In the yeast two-hybrid screening of several P.sojae RXLR effectors' interaction proteins in soybean,it was found that a cyclophilin protein GmCYP1 exists in the interaction protein candidate list of Avr3b,Avrlk and Avh23.Through one-to-one interaction verification,we confirmed that Avr3b,Avr1k and Avh23 can interact with GmCYP1 in yeast.It has been reported that the Glycine-Proline-Any-Leucine(GPxL)motif is the key region of interaction between cyclophilin and its substrate effector,and the GP motif is usually the binding site.Sequence analysis revealed that Avr3b,Avrlk,and Avh23 all contained GPxL sequences or the key GP sequences.Further analysis of 380 RXLR effector sequences from P.sojae found that 6 effectors contained the standard GPxL motif and 72 effectors contained the GP motif including Avh94.These effectors containing GPxL or GP motifs are all potential targets for the interaction of cyclophilin and may utilize the enzymatic activity of the plant's cyclophilin to activate its function in infection.Taken together,this study revealed a potentially mechanism and common strategy that the recruitment of plant cyclophilin protein are required for the activation of many RXLR effectors.