Identification And Characterization Of Receptor Like Kinase SOBIR1 Interactors

Rceptors that localize to host cell surface and in cytoplasm are ubiquitous and play an important role in immunity of plants to microbial pathogens upon recognition of pathogen. The cell surface receptors contain receptor like kinase(RLKs) and receptor like protein(RLPs). RLKs contain an extracellular domain involved in pathogen perception, a transmembrane domain and a cytoplasmic kinase domain for cellular signlling. In contrast to RLKs, RLPs lack a cytoplasmic kinase domain. The Cf proteins are RLPs mediating resistance to the fungal pathogens Cladosporium fulvum and the mechanism by how they trigger immunity remains enigmatic.Recently, we found that the RLK SOBIR1 can constitutively interacts with Cfs in plant and is required for Cf-mediated immunity. Furthermore, silencing SOBIR1 in N.benthamiana reduced the accumulation and stabilization of Cf-4. We and other researchers showed that SOBIR1 also interact with a broad range of RLPs.We proposed to understand how RLP complex mediates downstream signaling and what the roles of SOBIR1 in RLP-mediated immunity are by identifying its interactors. In this study, we identified the candidate interactors of SOBIR1 by yeast two hybrids and immunoprecipitation. We tested whether the Cf-4/Avr4 induced HR and resistance are affected after silencing candidate genes. The main results are as follows:(1)We performed a sequence alignment of Sl SOBIR1 and At SOBIR1 and found thatboth Sl SOBIR1 and At SOBIR1 have five predicted lucine rich repeats(LRRS), a signletransmembrane(TM) domain and a cytoplasmic kinase domain. From the alignments, thecytoplasimic kinase domain are more conserved than extracellular domain, and a conservedWxx Gxxx Gxxx G motif present in all aligned SOBIR1 homologs.(2)The construct PGBKT7-Sl SOBIR1-Kinase were made and transformed into yeast.The western blot results showed that SOBIR1-Kinase expressed in yeast. Further tests showedthe kinase domain of SOBIR1 did not have autoactivity and toxic. Then the kinase domain ofSOBIR1 was subjected to a yeast two hybrids to identify interactors of SOBIR1. VDAC andSAP18 were selected as SOBIR1 candicate interactors.(3)The constructs TRV-VDAC and TRV-SAP18 were made to target VDAC homologsand SAP18 homologs in N.benthamiana. SAP18 is required for Cf-4-mediated hypersensitiveresponse(HR), whereas VDAC not. Furthermore, CO-IP showed that VDAC can not beco-purified with SOBIR1.(4)We got transgenic tomato plants expressing 35S:SOBIR1-e GFP or 35S:FLS2-GFP confirmed by PCR and western blot. However, the efficiency of SOBIR1 transformation and expression are lower than FLS2 transformation, and the stable expressing SOBIR1 plant is dwarf.(5)Avr4 protein triggered Moneymaker-Cf-4 cell death. Dilution experiments showed when the concentration of Avr4 protein is higher than 100μg/ml, the HR was visible. Furthermore, the Avr4 protein at the concentration 1000μg/ml was infiltrated into the transgenic N.benthamiana expressing Cf-4, the clear cell death were observed. All the results indicated the Avr4 protein can induce Cf-4 mediated downstream signalling.(6)SOBIR1-GFP or SOBIR1-YFP protein were immunoprecipitated from transgenic tomato plants or transgenic Arabidopsis plants using GFP affinity beads. After transiently expressing SOBIR1-eGFP or SOBIR1-kinase-e GFP in N.benthamiana, the proteins were immunoprecipitated. DRP2, PP2 C, GB and STK were selected as SOBIR1 candidate interactors after mass spectrometry.(7)TRV constructs were made. After silencing exprements in N.benthamiana, STK was found to be required for Cf-4 mediated HR, whereas the rest genes seemed not to be required.