Functional Analysis Of Post-Translation Regulators ESD4 And SNIPER10 In Plant Immunity

Both small ubiquitin-like modifier（SUMO）-protease system（SPS）and Ubiquitin-proteasome system（UPS）play important roles in post-translational regulation to maintain proteins dynamic balance in plants.SPS involves linking SUMO to the target protein through a series of enzymatic reactions to affect the stability,activity and sub-localization of target protein.Whereas,UPS refers to the ubiquitination and degradation of target protein.In this study,a SUMO protease,ESD4 was found to involve in regulation of plant immunity through a forward genetics screening;While a ubiquitin ligase SNIPER10 was identified as an important regulator of plant immunity from a reverse genetics screening.The main results are as follows:1.When CRISPR-Cas9 system was used to knockout tandem RLP genes,a mutant line1-12 was identified.It exhibited constitutively activated defense phenotypes including dwarfism and,heightened expression of PR1/2,enhanced programmed cell death,increased accumulation of H₂O₂and increased resistance to H.a.Noco2.Map-based cloning and whole genome re-sequencing demonstrated that the phenotypes of line1-12 was caused by the loss function of ESD4.2.Compared with wild-type plants,q RT-PCR showed that the SA and NHP biosynthesis-related genes were highly expressed in esd4 mutant.It is speculated that ESD4 negatively regulates the plant immune response via inhibiting the accumulation of SA and NHP.3.EDS1 and NDR1 are two key regulators downstream of NLR proteins.Compared with esd4-3 single mutant,the autoimmune phenotypes were reduced in double mutants eds1-2eds4-3 and ndr1-1 esd4-3.The double mutants showed larger size with more flat leaves,reduced expressions of PR1/2 and SA/NHP biosynthesis-related genes,and decreased resistance to H.a.Noco2.These results indicated that ESD4 negatively regulates NLR-mediated immunity.4.Using Western blot assays,we demonstrated that loss of EDS1,NDR1 and FMO1reduced the accumulation of SUMO-conjugates in esd4 mutant.Additionally,exogenous application of NHP increased the accumulation of SUMO-conjugates in wild-type Col-0 plants.These data indicated that the ESD4-mediated Sumoylation is close related plant immunity.5.Overexpression of SNIPER10 enhanced the autoimmune phenotype of snc1 and snc2-1D,including dwarfism,increased PR1/2 gene expression,serious programmed cell death（PCD）and accumulation of H₂O₂.When overexpress SNIPER10 in Col-0,transgenic plants showed slightly autoimmune phenotypes.Compared with wild-type plants,the transcriptional expression of defense-related genes including SARD1,CBP60G,ICS1,PBS3,ALD1 and FMO1 were highly elevated in SNIPER10 overexpressed plants.These data indicate that SNIPER10 postively regulates plant immunity.6.Through protein sequence alignment,a homologous gene of SNIPER10 was identified and named as SNIPER11.Overexpression of SNIPER11 into snc1 also enhanced autoimmune phenotype of snc1.Pathogen infection assays showed that neither sniper10 or sniper11 single mutant showed significant difference in resistance to H.a.Noco2 and P.s.t DC3000 compared with wild-type plants.However,sniper10sniper11 double mutant showed enhanced susceptibility to those pathogens.These data indicated that SNIPER10 and SNIPER11 have functional redundancy in promoting plant immunity.7.Subcellular localization assay showed both SNIPER10 and SNIPER11 only expressed in nucleus;Tissue specific expression experiment indicated that SNIPER10 mainly expressed in roots,true leaves,petal and anther.SNIPER11 only slightly expressed in roots and anther.In addition,in vitro ubiquitination assay showed both SNIPER10 and SNIPER11 have in vitro self-ubiquitination activity and they are indeed functional E3 ligases.8.When overexpressed SNIPER10 into eds1-2 and ndr1-1 mutants,the autoimmunity caused by SNIPER10 overexpression was inhibited in varying levels,indicating that SNIPER10 positively regulates NLR-mediated immunity.The positive regulatory function of SNIPER10 on plant immunity partially depends on the activities of EDS1 and NDR1.This study enriched the content of plant immunity and provided theoretical reference for plant disease resistance genetic breeding.