Study On The Key Sites Of Cellulase EG1 Elicitor Function In Rhizoctonia Solani And The Function Of Its Interacting Protein

Rhizoctonia solani Kühn is a kind of plant soil-borne necrotrophic pathogenic fungus which causes sheath blight on maize and rice,resulting in necrotic spots on leaves,leaf sheaths and stems,seriously affecting the quality and yield of crops.Rhizoctonia solani can release a variety of cell wall-degrading enzymes during the infection of plants.Previous studies have shown that the?-1,4 endocellulase EG1 secreted by the necrotrophic pathogen Rhizoctonia solani can induce allergic necrosis,reactive oxygen species burst,Ca²⁺accumulation,extracellular alkalinization,callose accumulation,and protection related gene up-regulated expression,which behave as a typical excitor.In this study,the endocellulase EG1 was used as the research object,on the basis of the previous study that EG1 could induce plant immunity an acted as an elicitor,we identified the key segments and sites of its eliciting function according to transient expression in Nicotiana benthamiana and stable expression in yeast,and we screened a transaminase,ZmGABA-T(?-aminobutyric acid-transaminase),that involved in metabolic processes in Zea mays according to yeast two-hybrid assay,analysising of the interaction between pathogens and plants during necrotrophic pathogenic fungi infecting plants.We obtained two main results as following:1.The key regions and sites that EG1 induces plant immunity were identified.We removed the first 60 amino acids from the N-terminal on the basis of conserving the signal peptide of EG1,the truncated product of EG1,EG1-1,could still induce allergic necrosis and reactive oxygen species burst in Nicotiana benthamiana,besides this,EG1-1expressed and purified by Pichia pastoris GS115 could induce allergic necrosis and up-regulated of defense response-related genes when inoculated in vitro.The EG1-2,whose N-terminal 61-67 peptide(SPWAVND)were deleted compared to EG1-1,and the EG1-3,whose C-terminal 203-207 peptide(GCSRK)were deleted compared to EG1-1 could neither cause allergic necrosis,reactive oxygen species burst nor up-regulated expression of defense response-related genes in plants.Then we mutated the 61-67 peptide SPWAVND in N-terminal to alanine respectively,among them,P62A,W63A and D87A lost the ability to cause allergic necrosis of Nicotiana benthamiana leaves.However,when the 203-207 peptide GCSRK in C-terminal were mutated to alanine respectively,all of the mutants could induce allergic necrosis and the elicitor function were not affected.But when the C204 and R206were both mutated to alanine at the same time,neither transient expression could cause allergic necrosis in Nicotiana benthamiana leaves,nor in vitro inoculation with stably expressed purified enzyme could induce allergic necrosis in Zea mays and Arabidopsis thaliana leaves,the defense response-related genes were not up-regulated.The above results showed that the key regions of EG1 to induce plant immunity include two parts:SPWAVND in 61-67 regions and GCSRK in 203-207 regions.The key sites to induce plant immunity include:proline at position 62,tryptophan at position 63,aspartic acid at position 67,cysteine at position 204 and arginine at position 206,of which cysteine at position 204 and arginine at position 206 must exist at least one to ensure the normal function of EG1 elicitor function.2.The protein interacts with EG1 in Zea mays and its function in resisting R.solani infection has been determined.We found that EG1 was localized on the cell membrane in Nicotiana benthamiana according to confocal microscope observation.We used virus-induced gene silencing to silence the pattern recognition receptors BAK1 and SOBIR1 in N.benthamiana and found that transient expression of EG1 could still cause allergic necrosis in silenced leaves,this proved that EG1 inducing plant immunity was independent of the receptor kinases,BAK1 and SOBIR1.A?-aminobutyric acid transaminase ZmGABA-T(?-aminobutyric acid-transaminase)in Zea maize involved in glutamate metabolism and tricarboxylic acid cycle,which can convert the substrate?-aminobutyric acid GABA(?-aminobutyric acid)into succinic semialdehyde,and finally participate in the tricarboxylic acid cycle,was screened by yeast two-hybrid assay.Bimolecular fluorescence complementation(Bifc)and co-immunoprecipitation methods(Co-IP)proved that ZmGABA-T could interact with cellulase EG1 in plants,and the two co-localized on the cell membrane.When the EG1 and ZmGABA-T were coexpressed,we found that ZmGABA-T could inhibit EG1-induced allergic necrosis and reactive oxygen species(ROS)burst.When the ZmGABA-T was added into the EG1enzymatic reaction system,and the cellulase activity of EG1 was measured,the results showed that ZmGABA-T did not affect the activity of EG1.The substrate of ZmGABA-T,?-aminobutyric acid,GABA,was added into the culture medium of Rhizoctonia solani.And the transcription level of EG1 was detected,it was found that GABA could inhibit the transcription of EG1,but couldn't affect the enzymatic activity of EG1.When Rhizoctonia solani was inoculated on N.benthamiana leaves that transiently expressed ZmGABA-T,the size of the lesions caused by Rhizoctonia solani infection was suppressed,and the biomass of the pathogen decreased.Analysis and comparison of GABA-T sequences in different species found that GABA-T sequences are relatively conserved among different species,with differences only at the N-terminus.Only OsGABA-T in Oryza sativa could also interact with EG1 and inhibit the allergic necrosis of Nicotiana benthamiana leaves induced by EG1.Rice strains whose OsGABA-T were knocked out by CRISPR/Cas9 showed more larger lesion lengh and increased biomass of pathogens than wild type when infected with Rhizoctonia solani.The above results show that GABA-T in Zea maize and Oryza sativa can interact with cellulase EG1.GABA-T and its substrate GABA inhibit allergic necrosis and reactive oxygen species burst induced by EG1 according to promoting the degradation of EG1 and inhibiting the transcription of EG1 without affecting the activity of EG1,respectively.Thereby,the infection of necrotrophic pathogenic fungi,Rhizoctonia solani,was suppressed.These proved that the transaminase GABA-T involved in the glutamate metabolism process is also involved in the plant disease resistance process.In conclusion,our study proves that EG1 induce plant immunity including two segments and different sites,and transaminase in plants can interact with EG1 and participate in plant disease resistance.