Functional Analyses Of Tomato Calmodulin And Calmodulin-like Genes In Disease Resistance

Calmodulin (CaM) is a type of important Ca2+-binding protein. As the most important receptor of Ca2+, it is widely distributed in eukaryotes. CaM sequences are highly conserved in eukaryotes. The phylogenetic relationship of CaMs in some animals has been reported. However, that in plants remains unclear. Additionally, CaM plays crucial roles in plant growth, stress tolerance and disease resistance. However, little is known about the role and mechanisms of CaM in disease resistance in many economically important crops such as tomato (Solarium lycopersicum). In this thesis study, we focused on the role and mechanisms of CaM in disease resistance in tomato. The phenomenon that multiple CaM genes from a species or several species from diverse families encoded an identical CaM protein was unraveled. The phylogeny of CaMs in plants was elucidated. Moreover, the role and mechanisms of CaM in disease resistance in plants were revealed. The main results are as follows:(1) A total of150CaM genes were identified in37plant species from5genetic lineages, including higher flowering dicots and monocots, lower vascular non-flowering pteridophytes, nonvascular mosses and unicellular algae, and the phylogeny of CaMs in plants was revealed. Comparison between the CaM genes and their translated proteins demonstrated that the150plant CaM genes just encoded44proteins, which indicates that multiple CaM genes from a species or even several species from diverse families encoded an identical CaM protein. This phenomenon occurred widely in plants. All44CaM proteins contained four EF-hand motifs and had a size of149amino acids. Among them,13CaMs exhibited amino acid substitution in the supposed Ca2+-binding sites. Notably,6of them showed substitution with distinct amino acids. These CaMs included Pro33-OlCaM1from Ostreococcus lucimarinus, Pro34from Physcomitrella patens, Pro27-SmCaM2from Selaginella moellendorffii, Pro5-NbCaM7from Nicotiana benthamiana, Pro6-CpCaM12from papaya (Carica papaya) and Pro43-FvCaM2from wild strawberry (Fragaria vesca). The Ca2+-binding activity of these proteins awaited experimental verification. The phylogenetic tree of CaM proteins displayed that the CaM proteins clustered into three groups (group Ⅰ-Ⅲ). Group1was just comprised of CaMs from higher plants. Group Ⅱsolely contained CaMs from lower mosses and ferns, while group III consisted of CaMs from lower algae and bryophytes and higher dicotyledonous plants. Analysis of gene structure demonstrated that both group I and group Ⅱ CaM genes carried one intron, meanwhile, group Ⅲa did not contain intron. While group Ⅲb possessed three introns with a phase of1-0-0. Therefore, CaMs of group Ⅲb may be newly evolved. In addition, the Glycine residue at position60was substituted for Glutamine uniquely in group Illb CaMs. Whether this affects function of these CaMs awaits experimental evidence.(2) The role of CaM genes from plants including tomato in disease resistance was revealed. The expression pattern of various CaM and CML genes of Prunus persica and Nicotiana benthamiana in response to diverse pathogens was analyzed. The results showed that expression of PpaCaM in response to Taphrina deformans, that of NbCaMs to three pathogens, including rice bacterial blight pathogen(Xanthomonas oryzae pv. oryzae, Xoo), tomato leaf speckle pathogen(Pseudomonas syringae pv. tomato DC3000, Pst DC3000) and plant white mould pathogen(Sclerotinia sclerotiorum), was quite different. This indicated that different CaM genes might play different functions. VIGS silencing of SlCaM3and SICaM5resulted in highly accumulation of TRV and severe viral symptoms, and significantly reduced resistance to S. sclerotiorum. Silencing of SlCaM3, SlCaM4and SlCaM5decreased resistance to Pythium aphanidermatum; while transient overexpression of SlCaM3and SICML enhanced resistance to S. sclerotiorum. These results indicate that SlCaM3and SlCaM5play a positive role in resistance to TRV, S. sclerotiorum and P. aphanidermatum, and SlCaM4positively regulates resistance to P. aphanidermatum, while SICML plays a positive role in resistance to S. sclerotiorum.(3) The mechanisms of CaMs to regulate plant disease resistance were unveiled. Real-time PCR analysis showed that silencing of SlCaM3and SlCaM5strongly reduced the expression of PR1and PR5. Transient overexpression of SlCaM3and SICML decreased expression of Ca2+signaling genes CNGC17, CNGC18and CAMTA3. Results of yeast two-hybrid assay demonstrated that SlCaMs could interact with4SICAMTAs (S1CAMTA3/5/6/7) and5SICNGCs (S1CNGC5/15/16/17/18), among which, SICAMTA3and SICNGC16/17/18have been proved to be negative regulators of tomato disease resistance. Therefore, SICaM may function through interacting with SICAMTAs and SICNGCs and repressing their negative resistance regulating functions thereby promotes disease resistance. Moreover, employing prokaryotic expression and column affinity chromatography techniques, purified CaM6protein was obtained. Infiltration of2.5μM S1CaM6induced severe necrosis in leaves of N. benthamiana and rapeseed plants. Infiltration of1μM S1CaM6resulted in accumulation of reactive oxygen species (ROS). These results reveal that S1CaM6may enhance the resistance by inducing ROS accumulation and the hypersensitive response (HR).