Physiological And Biochemical Mechanisms Of Chitin Receptor-mediated Plant Immunity

In agriculture,the major plant pathogens that cause crop diseases are fungi.Most pathogenic fungi can cause necrosis,rot or wilting in plants.Plants have evolved complex immune defenses system to fight against pathogenic invasion.The plant defense response generally consists of two layers,i.e.,Pattern-Triggered immunity?PTI?and the Effector-Triggered Immunity?ETI?.The first layer of immune response is mediated by pattern recognition receptors?PRRs?located on plasmic membranes to activate PTI through extracellular sensing of Microbe-associated molecular patterns?MAMPs?.Chitin,the polymer of?-1,4-N-acetyl-glucosamine,is a major component of the fungal cell wall working as a typical MAMP triggering defense signals in plants.In previous studies,we found that chitin oligosaccharides?with degree of polymer less than 6,dp<6?is unable to trigger immune response or the interaction between At LYK5 and At CERK1,while,chitin oligosaccharides?dp?6?are strong elicitors that induce At LYK5 and At CERK1 interaction.Among the chitin oligosaccharides,chitin octamer is one of the best one to triggering plant immunity.At CERK1 and At LYK5 were experimentally shown to be the major chitin receptors in Arabidopsis thaliana.In A.thaliana,At LYK5 was proposed to be primarily responsible for the recognition of chitin,and At CERK1 form a complex with At LYK5 to and transmit signals downstream through its active kinase domain.Phylogenetic analysis showed that At LYK5 and At LYK4 are in the same branch,suggesting that they might share functional similarity.Indeed,the lyk4/lyk5 double mutant plants show no response to chitin.The miture of chitin polymer bought from Sigma and chitooctaose are often used in study to trigger plant defense.In this study,we compared the difference of plant immunity triggered by chitin mixture and chitooctaose in A.thaliana.as well as the function of LYKs proteins.The main finding inculdes:1. Both chitin mixture and chitooctaose induce the phosphorylation of CERK1determined as a band shift of CERK1 protein on SDS-PAGE gel.Both elictors induce strong CERK1 phosphorylation between 15 and 30 min.At the time point of 60 min,chitooctaose-induced CERK1 phosphorylation was lost,while chitin mixture-induced CERK1 phosphorylation was still detectable.These data indicate that chitin mixture and chitooctaose have functional difference in triggering plant immunity.2. There are five LYKs protein in Arabidpsis.To explore whether other LYKs are involved in chitin response.We constructed different Split LUC vectors that were transiently expressed in Nicotiana benthamiana to measure the interation between LYKs proteins.We observed that LYK4 interacts with LYK4 and LYK3 but not LYK3.Because LYK4 is the closest homolog of LYK5 and has function redundance with LYK5,it is possible that LYK4 forms a protein complx with LYK5 and CERK1 to mediate chitin perception.3. Transient expression of CERK1 triggers strong cell death in Nicotiana benthamiana,however,kinase-dead version of CERK1 does not induce a strong cell death phenotype in N.benthamiana,indicating that the kinase activit cell death.Mutiple published papers showed chitin induces a strong interaction between CEK1 and LYK5,however,we did not observed strong fluorescence signal kinase dead CERK1^K350N and LYK5 transiently expressed in N.benthamiana,suggesting that kinase activity of CERK1 is essential for interaction with LYK5.4. To investigate the function of the LYK5 intracellular domain,A.thaliana plants expressing LYK5-Myc was treated with chitin.The receptor complexes were obtained by affinity purification,and ATL6 and PUB38 were identified with mass spectrometry proteomic analysis.Both ATL6 and PUB38 were shown to interact with LYK5 in N.benthamiana using bimolecular fluorescence complementation and immunoprecipitation sassays.The interactions were induced after chitin treatment.However,ATL6 and PUB38do not interact with CERK1.We detected by Western blot and found that when ATL6 and LYK5 were co-expressed and treated with chitin,the protein content was less than that without chitin treatment;ATL6 and CERK1 were co-expressed and treated with chitin,the protein content was much less than that without chitin treatment.This indicates that ATL6inhibits the protein content of LYK5 and CERK1 after chitin treatment.5. We detected a small band below the full length of the LYK5 band using Western Blot,which is equivalent to the size of LYK5 kinase domain.To determine whether the band contains transmembrane domain or not,protein sample were purified with a nuclear protein purification kit.We found that the band is present in the supernatant suggesting that the protein was localized to the membrane.In summary,we systematically studied the interaction relationship between the Arabidopsis LYK receptor proteins,isolated and identified the LYK5 interaction proteins ATL6 and PUB38,and preliminary studied the possibility of regulation by mechanism of shear degradation within the intracellular domain of LYK5,in order to provide essential theoretical basis for further elucidating the molecular mechanism of chitin stimulating plant immune response.