Functional Analyses Of A Dual-Specificity Phosphatase, IBR5, In Temperature-Dependent Defense Responses In Arabidopsis

Plants have developed a complex and high-efficiency system to protect against microbial infection. R (Resistance) proteins play important roles in activation of ETI (Effector-Triggered Immunity) through recognizing specific effectors from microbe. Low temperature is an important environmental factor which adversely affects the production and quality of crops. Plants’ responses to low temperatures are tightly linked with defense responses. Some R proteins could be activated under low temperatures, such as SNC1 and RPP4 which belong to TIR-NB-LRR (Toll and Interleukin 1 Receptor-Nucleotide Binding-Leucine-Rich Repeat)-type R proteins. They are at inactive state under normal conditions. However, a specific point mutation in NB domain activates SNC1 or RPP4 protein under different temperatures even without pathogen attacking.Previous study in our lab has reported a chilling sensitive mutant, chs3-1, and the corresponding CHS3 gene. CHS3 encodes an atypical TIR-NB-LRR-type R protein with an additional LIM (Lin-11, Isl-1 and Mec-3) domain at its C terminus. The mutation in LIM domain results in abnormal splicing and a truncated protein. The chs3-1 gain-of-function mutant exhibits constitutively activated defense responses at 16℃, with small and curly leaves, extensive cell death, high SA (Salicylic Acid) and hydrogen peroxide accumulation as well as elevated expression of PR (Pathogenesis-Related) genes. Genetic analysis showed that the chilling sensitive phenotypes of chs3-1 is dependent on EDS1 (Enhanced Disease Susceptibility 1), SGT1b (Suppressor of the G2 allele of skpl) and RAR1 (Required for MLA12 resistance 1), which are required for the function of some R proteins.To further study the molecular regulatory mechanism of the temperature-dependent defense responses mediated by CHS3, we screened the suppressors of chs3-1 from EMS (ethyl methylsulfonate)-mutagenized chs3-1 mutant pools and identificated a suppressor of chs3, ibr5-7 (Indole-3-butyric acid response 5), which largely suppressed chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. Previous study has shown that ibr5 displays reduced insensitivity to auxin and ABA. Furthermore, MPK12 (Mitogen-Activated Protein Kinase 12) is shown as a substrate of IBR5 to negatively regulate auxin signaling but not to participate in the ABA-related phenotypes of ibr5 mutants. In this study, we showed that the regulation of CHS3 by IBR5 is also independent of MPK12. Biochemical studies revealed that IBR5 interacted with the TIR domain of CHS3 in vitro and in vivo. The mutated form of the IBR5 in catalytic motif did not affect their interaction, but only partially rescue the chs3 ibr5 mutant phenotypes, suggesting that the catalytic activity of IBR5 is required for full function in the CHS3-mediated defense response. Moreover, the accumulation of the CHS3 at mRNA and protein levels at chilling temperatures was inhibited by IBR5 mutation, suggesting that IBR5 is required for the accumulation of CHS3 protein, which might be at least partially attribute to increased CHS3 expression.In addition, we found that hsp90.3 is another suppressor of chs3-1. Given that sgtlb and rar1-20 also rescue the phenotype of chs3-1, HSP90-SGT1b-RAR1 complex may positively regulate CHS3-mediated immunity responses. In consistent with this notion, we found that IBR5 associated with HSP90 and SGTlb proteins in vivo. Furthermore, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Combined with the results of interaction between IBR5 with CHS3, these results suggest that IBR5 can form a complex with chaperone proteins, including HSP90 and SGTlb, to protect CHS3.In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also required for defense responses mediated by several other R genes. ibr5 partially inhibits the cell death and PR gene expression of bal and bonl-1 at 22℃. The immunity phenotypes of bal and bon1-1 are caused by activated SNC1 (Suppressor of nprl-1, Constitutive 1). IBR5 interacts with SNC1 in planta, suggesting IBR5 may regulate SNC1 activity directly.In addition, IBR5 is also required for RPM1 (Resistance to Pseudomonas Maculicola 1)- and RPS4 (Resistant to Pseudomonas Syringae 4)-mediated disease resistance. Biochemical evidence showed the interaction between IBR5 and RPS4 in vivo. Thus, this study uncovers the role of IBR5 in regulating multiple R protein-mediated defense responses, and sheds new light on the molecular mechanism and crosstalk of plant response to low temperatures and pathogen.