Regulation Of Tomato Responses To Alternaria Alternata F.Sp. Lycopersici And AAL-toxin By Plant Hormones

Tomato (Solanum lycopersicum) is one of the most popular and widely consumed vegetable crops in the world. Fungal diseases are the major factors, which limit crop production for their negative effect on yield and quality. Tomato production is affected by dozens of fungal diseases. Jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are conventional defensive phytohormones and play important roles in plant defense response against different biotic and abiotic stresses. ROS acting as signal molecules extensively participate in plant defense response against various stresses. With the progress of genetic research and tomato genome sequencing from International Solanaceae Genomics Project, tomato has become an excellent model system to study the cross-talk between plant and pathogen infection. In this study, the regulation mechanisms of JA, ET, and SA in stress tolerance and the cross-talk between them were investigated in the system of tomato-Alternaria alternata f. sp. lycopersici (AAL)-AAL-toxin. The results are as follows:1. The role of jasmonic acid (JA) signal in programmed cell death (PCD) induced by AAL-toxin in detached tomato leaves was analyzed using a 35S::prosystemin transgenic line (35S::prosys), a JA-deficient mutant spr2 and a JA-insensitive mutant jail. The results indicated that JA biosynthesis and signaling play a positive role in AAL-toxin-induced PCD process. In addition, exogenous application of ET action inhibitor, silver thiosulphate (STS) greatly suppressed the necrotic lesions in 35S::prosys leaves, although 35S::prosys leaflets co-treated with AAL-toxin and STS still have a significant high relative conductivity. Application of 1-aminocyclopropane-1-carboxylic acid (ACC) markedly enhanced the sensitivity of spr2 and jail mutants to the toxin. However, compared to AAL-toxin treatment alone, exogenous application of JA to the ET-insensitive mutant Never ripe (Nr) did not alter AAL-toxin-induced cell death. In addition, the reduced ET-mediated gene expression in jail leaves was restored by co-treatment with ACC and AAL-toxin. Furthermore, JA treatment restored the decreased expression of ET-biosynthetic genes but not ET-responsive genes in Nr mutant compared to the toxin treatment alone. Based on these results we suggest that both JA and ET promote the AAL-toxin-induced cell death separately, and the JAI1 receptor-dependent JA pathway also acts upstream of ET biosynthesis in AAL-toxin-triggered PCD.2. We have studied the role of H2O2 signal in JA-induced sensitivity of detached tomato leaves to AAL-toxin. The results demonstrated that the H2O2 content in jail leaves was significantly higher than that in CA leaves at 36 h after AAL-toxin treatment, however, the H2O2 content in jail was significantly lower than that in CA leaves at 48 and 72 h. We can concluded that the relatively low concentration of intracellular H2O2 at 36 h after toxin treatment may act as signal molecule and activate the defensive response, while the relatively high concentration of intracellular H2O2 after 36 h may cause severe damage to the plant tissues. O2-producing rate jail leaves was markedly lower than that in CA leaves at 36.48 and 72 h after AAL-toxin treatment, thus high level of toxic O2- molecules may cause serious damage to the plant tissues following AAL-toxin application. The activities of four antioxidant enzymes (SOD. CAT, APX and guaiacol POD) were studied in jail and wild type cv. CA leaves at 36,48 and 72 h after AAL-toxin treatment. The results showed that SOD and POD activity in jail was significantly higher than that in CA leaves at 36 and 72 h after toxin treatment, while APX and CAT activity mjail is significantly higher than that in CA at 36,48 and 72 h. These results demonstrated that compared with WT cv. CA, jail leaves may eliminate redundant ROS by inducing relatively high activities of APX, POD,CAT and SOD, thus reducing damage to leave tissues caused by AAL-toxin treatment. The expression patterns of five genes coding ROS metabolizing enzymes (NADPH Oxidase, Ch Cu. Zn-SOD, CAT2, APX1 and POD) in CA and jail leaves at different time points after AAL-toxin treatment were analyzed, and the mechanism of ROS pathway in JA-induced sensitivity of detached tomato leaves to AAL-toxin was elucidated.3. The role of ET signal in susceptibility to fungal AAL was investigated by utilizing ET overproducing mutant epi (epinaslic) combined with exogenous application of an ET precursor ACC, ET action inhibitor STS, and ET receptor inhibitor 1-MCP. The results showed that ET pathway plays a positive role in tomato susceptibility to fungal AAL through promoting fungal growth. The role of JA signal in susceptibility to toxin-producing fungal AAL was investigated by using JA mutants spr2 and jail as well as 35S::prosys transgenic line combined with exogenous MeJA application. We observed that JA biosynthesis and JAI1 receptor-regulated JA signaling pathway actually play a positive role in tomato susceptibility to fungal AAL through promoting fungal growth. In addition, the previous results of our group indicated that the expression of JA-related genes (LOXD, AOS2, and PI-â…¡) was down-regulated in jail leaves compared with CA at different time points after AAL infection. The role of SA signal in the interaction between AAL and tomato plants was also studied by using NahG transgenic line which has lower endogenous SA content combined with exogenous SA application, and the results suggested that SA signaling pathway might promote the resistance of tomato plants to fungal AAL through inhibiting fungal growth.The expression patterns of ET-related genes (ACO1, ACS4, ETR1, ETR3, and ERF1) and ET production in jail and CA leaves at different time points after AAL infection were analyzed. The results demonstrated that the expression of ET-related genes was down-regulated and ET production markedly decreased in jail leaves compared with CA at different time points after AAL infection, indicating that enhanced resistance to fungal AAL in jail plants was dependent on reduced ET signaling pathway. Subsequently, we pre-treated 35S::prosys plants with exogenous STS or pre-treated spr2 and jail plants with exogenous ACC respectively, and then inoculated the tomato plants with fungal AAL. The results demonstrated that blocking ET perception with STS pre-treatment markedly reduced the disease development in 35S::prosys plants. In addition, exogenous ACC application rescued the development of disease symptoms in spr1 plants and fungal growth in jail. In addition, we pre-treated exogenous MeJA to tomato plants with lower endogenous ET levels or inhibited ET reception, and then inoculated the tomato plants with fungal AAL. The results demonstrated that exogenous MeJA pre-treatment restored the development of disease symptoms in these plants, indicating that JA functions partly through ET pathway during the disease development process induced by AAL.STS pre-treatment markedly inhibited the development of disease symptoms in NahG plants, and SA pre-treatment did not change the the development of disease symptoms in epi plants, indicating that SA pathway might work upstream of ET pathway in this process.