Identification And Functional Analysis Of Suppressors Of Chilling Sensitive Mutant Rpp4-1d In Arabidopsis

Disease and extreme temperature are negative factors that affect the yield and quality of crops. During the long time competing with pathogens, plants have developed complicated mechanisms to defend themselves against disease. And this plant defense response can be regulated by temperature, an important factor of environment that affects plant growth and development. In the course of identifying pathogen, R proteins play an important role. Some R proteins can be activated under low temperature, such as SNC1in Arabidopsis and N protein in tobacco. A specific site mutations in NB-ARC domain of SNC1or N protein cause R proteins to be activated and the leaves to perform cell death at22℃. The CHS2/RPP4(Recogition of Hyaloperonospora Parasitica4) in Arabidopsis is also this kind of R protein. We found that a gain-of-function mutation in NB-ARC domain of RPP4leads to constitutive activation of the defense response under low temperature. And this rpp4-1d mutant exhibits chilling sensitive phenotypes, including curling, dwarfism, cell death, or even the death of whole plant.To further study the relationship between rpp4-mediated disease resistance and cold response, the suppressors of rpp4-1d was screened from an EMS mutagenesis library of rpp4-1d mutant. Suppressors rar1, sgt1b, hsp90.2and hsp90.3have been identified. The hsp90mutants largely suppress the activation of defense response and chilling sensitive phenotypes of rpp4-1d. The mutated hsp90could interfere with the wild-type HSP90in a dose-dependent manner. The hsp90mutants exhibited compromised RPM1(Resisance to Pseudomonas Maculcola1)-, RPS4(Resistant of Pseudomonas Syringae4)-and RPP4-mediated pathogen resistance, compared to the wild-type. However, the requirement degrees of HSP90in the pathogen resitance mediated by these R proteins are different.It has been reported that chaperonin complex RAR1-SGT1B-HSP90could interact with some R proteins to maintain the normal structures and physiological functions of proteins. Our study also proved the both wild-type RPP4and the mutated form rpp4can interact with HSP90to form protein complexes. And the formation of this complex could not be affected by low temperature. Further more, the stability of rpp4protein in hsp90mutant had not changed as well. RPP4and rpp4proteins were localized in both cytoplasm and nucleus under normal temperature, Nevertheless, not like RPP4, low temperature reduced the accumulation of rpp4protein in nucleus, which was not affected in hsp90mutants. Genetic analysis of the intra-genic suppressors of rpp4-1d revealed the auto-activation of TIR domain and the important functions of the NB-ARC and LRR domains of RPP4in temperature-dependent defense signaling. In addition, the chilling sensitivity of rpp4-1d was largely independent of the WRKY70or MOS (Modifier of snc1) genes.In summary, this study suggests that RPP4-mediated disease resistance and cold response both depend on HSP90proteins. HSP90and rpp4proteins form a complex in vivo. The mutated hsp90does not affect the stability and subcellular localization of rpp4protein. The mechanisms of rpp4and sncl-mediated responses are different. The chilling-sensitive cell death mediated by rpp4is similar to the hypersensitivity in disease resistance. The mutated hsp90protein has interfering effect on the wild-type HSP90, which not only inhibits rpp4-mediated chilling sensitive phenotypes, but also reduces R protein-mediated defense resistance. Our research has provided experimental evidence to the influence of temperature on disease resistance and a new clue to study various mechanisms of R protein-mediated disease resistance.