Molecular Genetic Analysis Of The Regulation Of Arabidopsis Immune Receptor SNC1

The recognition of pathogen effector by the disease resistance gene NB-LRR(nucleotide binding-leucine-rich repeat)in plant immune system is an effective way for plants to trigger immune response.SNC1(SUPPRESSOR OF nprl-1)is a TIR-type NB-LRR(Toll/interleukin-1 receptor-NB-LRR)disease resistance gene in Arabidopsis,and its expression level in plants as a marker of plant immunity,which is also correlated with plant growth and development.The enhanced immune response usually leads to defects in plant growth.Therefore,NB-LRR resistance genes need to be finely regulated in plants.Previous studies have shown that there are complex regulatory mechanisms for the expression of SNC1,including the regulation at transcriptional and post-transcriptional level.MOS1(modifier of sncl)can bind to the promoter region of SNC1 and positively regulate the expression of SNC1 at chromatin level.Therefore,in order to achieve the balance between plant disease resistance and growth,there must be negative regulation factors at chromatin level.The underlying factors is rarely reported and the molecular mechanism of SNC1 regulation at chromatin level is still unclear.NB-LRR is not only precisely regulated by plant endogenous regulators,but also the environmental factors can affect its activity and function.It has been found that an increase in ambient temperature inhibits the nuclear localization of NB-LRR,which in turn affects its activity,but the specific molecular mechanism is still unknown.Our study started from two aspects to explore the regulation mechanism of SNC1 by unknown regulators and temperature.First,the bonlmosl mutant was used as background to screen the mutants that restored bon1 phenotype and inhibited bonlmosl phenotype by EMS(ethyl methanesulfonate)mutagenesis.To detect the molecular mechanism of SNC1 regulation by mapping new regulators.By screening the growth phenotype of M2 plants after mutagenesis,we obtained a mutant with growth phenotype like bon1,named suppressor of bonlmosl,bonlmoslsmo.The growth phenotype of bonlmoslsmo dependent on bonlmosl rather than smo mutation resulting in a bon1-like phenotype.The disease resistance of bonlmoslsmo was significantly higher than bonlmosl and turned to the bon1 level.And the expression level of SNC1 in bonlmoslsmo is much higher than bon1mosl.After mapping by sequencing,we found that SMO is a ubiquitin-like superfamily protein,and function prediction says it may be involved in mRNA splicing.The subcellular localization determined that SMO localizes in the nucleus.Transcript expression analysis revealed that SMO does not regulate SNC1 expression by mRNA splicing.RNA-seq analysis showed a large number of genes involved in chromatin remodeling and histone modification are up regulated in smo mutants.Among them,chr5 and atxr7 can suppress the growth phenotype of bonlsmo,indicating that CHR5 and ATXR7 may act downstream of SMO.Compared with bonlmosl,in bonlmoslsmo,the enrichment of H3K4me3 and H2Bub1 in SNC1 promoter and coding region were significantly increased.Those results indicate that SMO may regulate the expression of SNC1 by chromotin remodeling and histone modification.Second,we used a variety of Arabidopsis nucleoporin loss of function mutants to mimic the nucleoporin complexes which is composed of different components in plants,then observed the phenotype of these mutants both in Col-0 and temperature-insensitive auto-immune mutants sncl-4 background.The observation of growth phenotype and disease resistance in different temperature showed the high temperature sensitivity of nucleoporin loss of function mutants.We hypothized high temperature may affect the nuclear accumulation of SNC1 through nucleoporin then affect plant immune response.The results showed some nup mutants such as nup96 and nup133 showed high temperature sensitivity in Col-0 or sncl-4,indicating the important role of nucleoporin in high temperature response.The transcriptomic analysis of nup96 mutant showed most of the genes regulated by nucleoporin were high temperature response genes,which further confirmed the importance of nucleoporin in high temperature response.It was also found that NUP85 and NUP133 are involved in regulating mRNA export only under high temperature conditions.We also found that nup mutants can completely suppress the auto-immune phenotype of sncl-4 only at high temperature,indicating that the presence of nucleoporin is required for the disease resistance of sncl-4 under high temperature condition,implying the accumulation of SNC1 in the nucleus depends on the composition of nucleoporin complex,which indirectly indicates that high temperature condition may alter the composition of the nucleoporin complex,thereby inhibit the nuclear accumulation of SNC1 and affect plant immune response.Our research not only found new regulatory factors involved in SNC1 expression,but also opened up a new insight for exploring SNC1 regulation mechanism at epigenetic level.We also gave a good explanation about the molecular mechanism of high temperature regulates plant immune response through SNC1 nuclear accumulation mediated by nucleoporin.It provides important information for excavating the regulation mechanism of SNC1 in plants.