Proteomics And Defense Mechanism Analysis Of An Arabidopsis Gain-of-resistance Mutant Cpr30

Much attention has always been paid to the molecular mechanisms of plant defense response against pathogen infection. Over the years, many new components involved in plant immunity have been elucidated, yet some joint points of the entire pathway of plant defense are still unclear. The mutants that exhibit native disease resistance are powerful materials to dissect the plant defense mechanisms. Furthermore, proteomic study on these materials can help us obtain systematical cognitions for protein expression profiles of the defense response involved in these mutants. The F-box protein CPR30is a negative regulator of plant defense response, and its loss-of-function mutant cpr30, which show HR-like lesion formation and SAR-like constitutive defense responses, is a typical gain-of-resistance mutant. In the present study, we analyzed the proteome of cpr30and explored its defense mechanism preliminarily.The total proteins of cpr30and the control (wild type) were separated by2-D electrophoresis, and analysis of the2-DE patterns revealed that the expression level of52proteins had been changed significantly in cpr30. Among the52spots digested with trypsin and identified by MALDI-TOF mass spectrometry,33spots, a major of which were strongly up-regulated and had sufficient abundance, obtained high and credible scores. The identified proteins were classified into five groups based on their putative functions, including pathogen-defense, redox homeostasis, energy metabolism, signal transduction, and protein folding and homeostasis. We compared the mRNA abundance of some genes encoding affected-proteins via quantitative RT-PCR to see if there are any changes between mRNA level and protein level, and the results showed that the protein level was not coincided with the mRNA level, which might be due to the posttranslational modification. We further confirmed the expression pattern of some important proteins via Western blot. Consistent with our2-DE results, Western blot analysis also indicated that the expressions of these proteins were indeed greatly affected in cpr30. In addition, the Western blot results also demonstrated that the2-DE analysis yielded fairly accurate measurement of protein expression.We also studied the proteome of the double mutant pad4cpr30, which almost completely suppressed the phenotype of cpr30. Nine protein spots whose abundance had changed significantly were identified by2-DE coupled with MALDI-TOF-MS. Function analysis indicated that these proteins were involved in protein folding, redox homeostasis, energy metabolism and amino acid metabolism. Six spots of which increased significantly in pad4cpr30were also increased in cpr30, indicating the possibility that they may involved in the defense pathway regulated by CPR30.The F-box protein CPR30can interact with ASK proteins to form SCF complex, so it might degrade some yet unknown substrates through ubiquitination, and the substrates might be one or more proteins that induced in cpr30. The yeast two-hybrid assays were performed to screen the substrates interacted with CPR30. The results showed that only SGTla and its homolog SGTlb could interact with CPR30. The function of SGT1a and SGTlb involved in cpr30were explored through the generation of the double mutants sgtla cpr30and sgtlb cpr30. The appearance of these double mutants indicated that sgtla and sgtlb partially suppressed the dwarf morphology of cpr30, as well as cell death and disease resistance conformed by typan blue stain and pathogen infection. Above all, these results suggested that SGTla and SGTlb played positive roles in the defense response regulated by CPR30.