| Plants always suffer a variety of stresses from their external environment. To survive, plants have developed complex mechanisms including a series of signal pathways to regulate the responses against these stresses. In this study, we isolated and identified four tomato gene families associated with plant signal transduction network, and analyzed their possible biological functions in responses to biotic and abiotic stresses.Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that mediate the transduction of extracellular stimuli via receptors/sensors into intracellular responses and play key roles in plant immunity against pathogen attack. However, the function of tomato MAPK kinases, SlMKKs, in resistance against Botrytis cinerea remains unclear yet. In order to study the possible roles of SIMEKs in tomato resistance to B. cinerea, we isolated and identified five tomato SlMKKs genes, which are homologous to Arabidopsis AtMEKs. After infection with B. cinerea or treatments with signaling molecules including salicylic acid, jasmonic acid or1-aminocyclopropane-1-carboxylic acid, these five SIMEK genes exhibited different expression patterns. The function of these SIMEK genes in resistance to B. cinerea was examined through silencing individual SIMEK gene using virus-induced gene silencing (VIGS) approach. VIGS results indicate that the SIMEK2-and SlMEK4-silenced plants showed more susceptibilities to B. cinerea, and the expression levels of four PR genes, SlPRlb, SlPRP2, SILapA and SIPIN2, were also significantly lower than that of nonsilenced control plants. Additionally, the accumulation of reactive oxygen species (ROS) induced by infection of B. cinerea in SlMEK2-and SlMKE4-silenced tomato plants were higher than that of in the nonsilenced control plants. On the other hand, when constitutively phosphomimicking forms of SlMEK2and SlMEK4, SlMEK2DD and SlMEK4DD, respectively, in Nicotiana benthamiana, occurrence of HR, accumulation of ROS, enhanced resistance against B. cinerea, and up-regulated expression of PR genes (e.g. NbPRl, NbPR2, NbPR4and NbPR5), were observed. Taken together, our results demonstrate that tomato SIMEK2and SIMKE4play important roles in resistance against B. cinerea.Calmodulin-binding transcription activators (also called SRs) are calmodulin-binding transcription factors, which play an important role in Ca2+signal pathway. Seven SR genes have been identified in tomato genome; however, the functions of tomato SlSRs family genes in biotic and abiotic stress response remain unclear. Expression of SlSRs genes were induced with different patterns after infection with B. cinerea or Pst DC3000. Silencing of SlSR1and SISR3L in tomato plants resulted in enhanced resistance against B. cinerea and Pst DC3000, spontaneous induction of ROS and up-regulation of defense-related genes (e.g. SlPR1b, SlPRP2, SIRbohl). Meanwhile, drought stress also induced significantlyexpression of SlSR1L. Further experiments revealed that silencing of SlSR1L reduced drought tolerance with accelerating water loss and down-regulated the expressions of drought related genes (e.g. SGN-213276, SlAREB1, SlAREB2, SlDREB and SlSpUSP). Transactivation analysis showed that both S1SR1and S1SR3L had transactivation activity and subcellular localization assays demonstrated that both the S1SR1and S1SR3L were nucleus-localized proteins. These results indicate that the tomato SlSRs participate have diverse functions in resistance against B. cinerea or Pst DC3000and in tolerance to drought stress.Plant Respiratory Burst Oxidase Homologues (RBOH) genes are one of major enzymatic sources of ROS in oxidative burst of plant cells challenged with pathogens or elicitors. The possible roles of tomato SIRbohs indisease resistance were studied. A total of eight SlRbohs genes were identified in the tomato genome and all these SIRboh genes encode typical NADPH oxidase proteins containing conserved motifs including EF-hands domain, FAD-bind domain, NADPH-binding domains. qRT-PCR results showed that the expressions of SIRbohs genes was differentially induced by infection with B. cinerea or Pst DC3000. VIGS results indicate that SlRbohB-silenced plants were more susceptible to B. cinerea when compared with the nonsilenced control plants. In SIRbohB-silenced tomato plants, the inductions of ROS and PR genes by infection of B. cinerea and treatment of flg22were restrained, suggesting that SIRbohB might be involved in the interaction between tomato and B. cinerea through medicating the generation of ROS at early stage of infection. By contrast, transient expression of SIRbohB in N. benthamiana resulted inenhanced resistance against B. cinerea but did not induce generation of ROS. Furthermore, the SIRbohB-silenced plants showed more susceptibility to drought stress, higher ratio of water loss under drought condition, and significantly down-regulated expression of SGN-213276and up-regulated expression of SGN-214777. The SIRbohB-GFP was localized to the membrane. These results demostrate that SIRbohB participates in responses to biotic and abiotic stresses.S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes, which plays an important role in plant growth and response to biotic and abiotic stresses. However, the functions of SISAHHs genes in tomato have not been elucidated. There are three SISAHHs members in tomato genome, and their amino acid sequences share more than97%of identity. We investigated the expression patterns of these three SISAHHs family genes after pathogens infection and exogenous hormones, SA, MeJA, and ACC treatments. The results showed that the expression of SlSAHH1was strongly induced by Pst DC3000whereas SlSAHH2and SlSAHH3were significantly induced by B. cinerea. Additionally, the expression of SlSAHH2was induced by all the three hormones, and SlSAHH3was induced by SA and MeJA. Silencing of SlSAHHI, SlSAHH2or SlSAHH3in tomato plants did not affect growthand development. However, combined silencing of SlSAHHI, SlSAHH2and SISAHH3together significalty affect growth and development of tomato plants such as stunting growth, curling leaf and fine root. Further studies suggested that silencing SlSAHHa plants could strongly induce the expressions of SA-related PR genes and accmulation of ROS. Furthermore, the SISAHHa-silenced plants showed increased resistance to Pst DC3000and enhanced tolerance to drought stress, but did not alter the resistance to B. cinerea. These data demonstrate that tomato SISAHHs genes play important roles in disease resistance and drought tolerance. |
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