Systematic Functional Characterization Of Leucine-Rich Repeat Receptor-Like Kinases(LRR-RLK) In Phytophthora Sojae

Phytophthora is an important class of plant pathogens belonging to the Oomycetes family(Stramenopiles).Due to the wide range of hosts,destructiveness and harmfulness,crop diseases caused by Phytophthora sojaeare devastating,posing a serious threat to global ecology and food security.Soybean root rot caused by Phytophthora sojae is one of the devastating diseases in soybean production and can infect soybeans at any stage,causing economic losses up to $2 billion worldwide annually.Phytophthora sojae enters different life cycles by recognizing changes of external environmental factors,sensing the host and secreting a large number of degradative enzymes,protease inhibitors,and effector proteins to complete the infestation process.Therefore,the analysis of how Phytophthora senses external signals and how the signal transductionoperate provides a theoretical basis for finding new fungicide targets.Leucine-rich repeat-like receptor kinases(LRR-RLKs)are key membrane receptor proteins in plants,which play a crucial role in signal recognition and communication between cells and between cells and environment.Approximately 60 LRRRLKs with reported biological functions play important roles in various developmental and physiological processes,such as plant growth and development,immune responses,and abiotic stresses.Like plants,the oomycete pathogen genome also contains LRR-RLKs,but their functions remain largely unknown.Considering the critical role of plant LRR-RLKs throughout the plant life cycle and in plant-environment interactions,deciphering the function of oomycete LRR-RLKs in host-oomycete interactions could help find new targets for oomycete control.Thus,this study systematically analyzed the function of LRR-RLK in Phytophthora sojae using CRISPR/Cas9 gene editing technology to reveal the functional diversity and redundancy of the LRR-RLK gene family in sporangium formation,chemotaxis,pathogenicity and stress response.Systemic functional analysis and identification of Ps RLKs in Phytophthora sojae:24 LRR-RLK genes(Ps RLKs)were identified in Phytophthora sojae by bioinformatics analysis.These Ps RLK proteins are structurally similar to plant LRR-RLK,with multiple LRR repeat sequences in the extracellular region,transmembrane domain and intracellular kinase domain.However,Ps RLK has a relatively short extracellular domain,a small number of LRR motifs,and a kinase region that evolves independently from plant kinases.To systematically analyze the function of Ps RLK,in this study,24 Ps RLK-knockout mutants were obtained by using CRISPR/Cas9 gene editing technology and their growth,resistance,zoospores production,chemotaxis,and infection were analyzed.All Ps RLK mutants did not affect mycelial growth rate,but four Ps RLK mutants(ΔPs RLK12/16/20/23)were more susceptible to the fungicide metalaxyl.Three Ps RLKs(Ps RLK2/8/18)were involved in the stress response to the Bacillus amyloliquefaciens FZB42,while the other three Ps RLKs(Ps RLK12/16/20)were involved in susceptibility to Pseudomonas fluorescens 2P24.RTPCR analysis showed that co-culture with FZB42 induced up-regulated expressions of Ps RLK8/12/18 genes,while Ps RLK12/16/20 genes were up-regulated upon incubation with2P24.Seven Ps RLKs(Ps RLK2/4/8/17/20/23/24)are important for zoospore production.The other five Ps RLKs(Ps RLK5/11/17/22/24)are involved in the chemotaxis of zoospores to isoflavones.Six mutants which Ps RLKs(ΔPs RLK8/12/15/18/19/20)rwere knockedout exhibited significantly reduced virulence,four of which(ΔPs RLK8/18/19/20)were defective in inhibiting host reactive oxygen species burst(ROS)was defective.Establishment of Ps RLK Interaction Network: To further explore the mechanism of Ps RLK signaling in Phytophthora,we screened Ps RLK interactions using the LCI interaction protein analysis method.We tested 576 Ps RLK pairs and found 64 positive reciprocal pairs.Of these,11 Ps RLK proteins(Ps RLK2/6/7/9/11/13/15/19/22/23/24)had no detectable interactions with other or themselves,and three Ps RLK proteins(Ps RLK10,Ps RLK14 and Ps RLK21)served as interacting nodes and were not found to have any function in previous studies.Thus,it was hypothesized that there might be functional redundancy in Ps RLK.We further obtained two mutantswithout double genes,ΔPs RLK21&10 and ΔPs RLK21&17,and phenotypic analysis revealed that ΔPs RLK21&10 sporulation was severely impaired and virulence was reduced.ΔPs RLK21&17 virulence was reduced and ROS staining revealed that its ability to inhibit host ROS was diminished.In contrast,ΔPs RLK21&17 showed an increased number of sporangia compared to the P6497 and ΔPs RLK21,with no significant difference compared to ΔPs RLK17.It is thus hypothesized that Ps RLK17 has dual roles in regulating the infestation process by interacting with Ps RLK21 and a signaling pathway that does not depend on Ps RLK21 to regulate sporangial production.The results of this study provide clues for later studies on the mechanisms of Ps RLK-regulated signaling pathways.The regulatory mechanism of Ps RLK and Ps GPA1: The chemotaxis of zoospores in Phytophthora sojae has been reported to be mainly regulated by the G protein signaling pathway.Silencing of Ps GPA1,a protein encoding the Gα subunit in Phytophthora sojae,resulted in loss of swimming spore tropism and frequent turning,and reduced virulence.This suggests that the transcriptional expression of Ps GPA1 affects the chemotaxis of zoospores.In our previous experiments,we found that five Ps RLKs(Ps RLK5/11/17/22/24)were involved in regulating zoospore chemotaxis,and in this study,we analyzed the transcriptional expression of Ps GPA1 in these five Ps RLKs knockout mutants at the zoospore stage by RTPCR.The sharp decrease of expression of Ps GPA1 was found in ΔPs RLK17/22/24,which suggested that Ps RLK may affects zoospore chemotaxis by regulating Ps GPA1.Immunoregulation by LRR-RLK in Arabidopsis through interactions with G proteins.This study further demonstrated that Ps RLK interacts with Ps GPA1 through the intracellular kinase region using LCI,Y2 H,and in vitro pull-down analysis.Analysis of phosphorylation in vitro revealed that Ps RLK5/11/17/22/24 kinase regions all have kinase activity and only Ps RLK11/24 could phosphorylate Ps GPA1.These implies that the regulatory mechanism of LRR-RLK and G proteins in plants is conserved in oomycete,laying the foundation for further elucidation of the chemotactic regulatory mechanism.This study discloses the functions of all LRR-RLK genes in Phytophthora sojae.The functional diversity and redundancy of the LRR-RLK gene family in sporangium formation,chemotaxis,pathogenicity and stress response were revealed.Ps RLK-Ps RLK interaction network was generated,which revealed that many Ps RLKs can form homodimers or heterodimers through their LRR domains and demonstrated that five LRR-RLK genes are involved in chemotaxis through interaction with the Gα protein Ps GPA1.This study reveals the diverse and important roles of a new gene family in oomycete pathogens,and resolves the regulatory mechanisms of LRR-RLK that may be involved in mobile spore chemotaxis,interaction with biocontrol agents and fungicide resistance to offerhelp in the study of the pathogenesis of Phytophthora and the development of new disease control strategies,as well as beinga starting point for understanding the functions and mechanisms of LRR-RLK in non-plant life forms.