In Arabidopsis leaves infected with the bacterial pathogen Pseudomonas syringae, the ET response factors EIN3 and EIL1 appear to negatively regulate plant immune responses by disrupting the pathogen-induced accumulation of SA . Thus, the decrease in LeEIL4 and LeEIL43 expression during fruit infection may represent a plant strategy to modulate the intensity of the ET response to B. cinerea, and/or to avoid the repression of SA biosynthesis. The expression of other ET signaling component genes also is enhanced during ripening, but specific expression changes after infection depend on the ripening stage of the fruit . For example, the protein kinase LeCTR4 is up-regulated in infected RR fruit, and LeERF1 expression increased in infected MG fruit but isreduced in infected RR fruit. Even though LeERF1 has been reported to induce fruit ripening and softening , its over-expression also is associated with resistance of RR tomato fruit to the necrotroph, Rhizopus nigricans . In addition, ERF1 serves as an intersection point between ET and JA response pathways triggering plant defenses, particularly against necrotrophs . By qRT-PCR no change in expression of LeERF1 was detected in infected RR fruit; therefore, further analyses using additional biological material, round pot including infections of fruit withother pathogens, are necessary to reliably assess the regulation of ERF1 expression in responses to infections.

Experimental observations have suggested that low concentrations of ET are required to induce defense responses in fruit prior to pathogen infection , while high and/or persistent ET levels have been related to increased pathogen susceptibility . ET production in fruit is considered to be under the control of two systems, designated Systems 1 and 2. The role of each system is specific to the plant species and developmental stage . System 1 is characterized by low levels of ET synthesis due to auto-inhibition and is present throughout early fruit development and during ripening of non-climacteric fruit . System 2 refers to the autocatalytic synthesis of ET that is active at the onset of ripening in climacteric fruit and that leads to high levels of accumulated hormone . It is possible that ET is generated in unripe fruit after pathogen recognition under System 1 and that this pathogen-induced concentration of ET specifically activates the expression of defense genes and/or other resistance pathways, but once the ET levels surpass a threshold, induction of System 2 and the associated climacteric ripening, or the activation of senescence/ripening pathways in non-climacteric fruit, may lead to enhanced susceptibility regardless of the defense mechanisms activated.

Therefore, ET can act as a promoter of susceptibility or resistance depending on its levels in the tissue and on the developmental stage of the host; in the case of fruit, this corresponds to the point at which the tissue is competent to respond to different ET concentrations. The hypothesis that ET responses during tomato fruit infection depend on the concentration and perception of this hormone is supported by the results shown in Figure 4. In this experiment, tomato fruit at MG and RR stages were pre-treated with either high levels of ET , or low or high levels of the ET inhibitor, 1-MCP, prior to inoculation with B. cinerea. 1-MCP, which disrupts ET responses by essentially irreversibly binding to the plant cell ET receptors and maintaining their phosphorylation state , has been widely used to study ripening and disease development in fruit . Pre-treatment of fruit with ET had no effect on infections of MG fruit by B. cinerea; these fruit were about to enter the climacteric phase of ripening and were capable of perceiving the hormone. Pre-treatment with ET also did not affect infections of RR fruit, which had already established ET-induced ripening processes. Pre-treatment with low levels of 1-MCP initially reduced infections in both MG and RR fruit; however, resistance was maintained only in MG fruit in which the climacteric increase of ET was delayed. Pre-treatment with high levels of 1-MCP prematurely induced susceptibility in MG fruit but did not influence RR fruit infections.

These observations suggest that low concentrations of 1-MCP may block some but not all ET receptors probably because of limited amounts of the inhibitor and continuing de novo generation of receptors. Thus, ET might be perceived in an appropriate concentration to promote resistance in the presence of low 1-MCP levels. In contrast, high 1-MCP levels may block ET perception longer and, thereby, hamper resistance response mechanisms that rely on ET perception. Previous studies also confirmed that application of high concentrations of 1-MCP prior to inoculation with other pathogens often induces rapid decomposition of climacteric and non-climacteric fruit, while application of low concentrations tends to reduce or stop infections . ET-mediated defenses are generally effective for controlling biotrophs, but are frequently inadequate against necrotrophs . Certain necrotrophic pathogens, such as Penicillium digitatum and B. cinerea, are capable of producing ET, possibly as a virulence factor and/or to induce ET synthesis in the host, thus promoting premature senescence or ripening . However, it is not possible to distinguish experimentally in infected tissues between the ET synthesized by the pathogen or by the host. While it is known that ET is synthesized by B. cinerea using the 2-keto- 4-methylthiobutyric acid pathway rather than the ACC pathway used in plants, the genes responsible for ET biosynthesis by B. cinerea have not been identified so inferences about total ET abundance based on biosynthetic gene expression of both organisms cannot be made yet. The dissimilar roles of ET in necrotrophic and biotrophic infections may relate to the model of ET concentration-dependent responses of plant tissues. Low levels of ET may effectively control both biotrophs and necrotrophs, but higher ET levels may favor only necrotrophic infections. Whether a pathogen is capable of perceiving ET and responding to the hormone during its development or when interacting with the host is also relevant in infections and should be explored further.Two routes of SA biosynthesis had been described in plants, the isochorismate pathway and the phenylalanine ammonialyase pathway, but neither pathway has been completely resolved . SA synthesis in response to pathogen infection and abiotic stress is apparently preferentially by the IC pathway , while the PAL pathway may have a minor contribution in local resistance . No significant changes in gene expression in either SA biosynthesis pathway were detected in the microarray analysis. Only the expression of WES1, a SA-modification enzyme, increased as consequence of ripening and infection, as shown in the microarray and validation studies . Further up-regulation of WES1 was also observed later in infection in both MG and RR fruit . WES1 catalyzes SA–Asp conjugation . The SA–Asp conjugate is considered to be an inactive form of SA and a target for catabolism . Thus, this result may suggest that SA inactivation occurs during fruit ripening and is a generalized response of tomato fruit to pathogen challenge regardless of the ripening stage. Moreover, SA can influence the levels of other hormones, including ET , round plastic planter and in fruit it could interfere with the regulation of ripening. Further characterization of the SA synthesis pathways and studies of the hormone’s production/modification during fruit development are needed to understand fully its impacts on fruit–pathogen interactions. SA signaling occurs via NPR1-dependent and -independent pathways .

NPR1 is a transcriptional co-regulator of SA responses and has been recently identified as a receptor of SA in plants . In the NPR1-dependent pathway, NPR1 monomers interact with members of the TGA family of bZIP transcription factors to regulate expression of SA responsive genes . TGA factors can be activators or repressors depending on the presence of SA and their ability to form specific protein complexes . From the microarray and qRTPCR results, the down-regulation of a tomato homolog of TGA6 in MG fruit and its up-regulation in RR fruit suggest that this gene may serve as a control point to modulate SA signaling during fruit–pathogen interactions . Tomato TGAs have been previously implicated in resistance against biotrophs and can be recruited by necrotrophic pathogens to induce susceptibility . Independently from NPR1, the protein kinases MAPK3 and MAPK6 have been shown to be important in systemic acquired resistance and priming for resistance . Pre-treatment with low concentration of SA prior to pathogen encounters induces the accumulation of inactive MAPK3 and MAPK6 in vegetative tissues and once an infection occurs, these kinases are rapidly activated to enhance the expression of defense genes . The phosphatases, PTP1 and MKP1, inactivate both MAPK3 and MAPK6 and therefore suppress the downstream SA signaling pathway . In infected fruit, a significant decrease in expression of a PTP1 homolog is observed only in resistant fruit, which may lead to the activation of the MAPKs. In particular, a tomato homolog of MAPK6 appears to be significantly up-regulated in MG fruit after B. cinerea inoculation . These results indicate that SA responses via the MAPK pathway may be distinct from those mediated by NPR1 and that these responses may be necessary for both basal and induced defenses in MG fruit. The susceptibility of the NahG tomato line, which does not accumulate SA , provides additional support for the hypothesis that some SA responses can contribute to resistance in fruit . When we inoculated NahG fruit with B. cinerea conidia, the fruit at the MG stage were significantly more susceptible to B. cinerea infection than their wild-type counterparts and did not generate the localized necrotic response surrounding the inoculation site that is common in resistant unripe fruit. The localized necrotic response in MG fruit is associated with an oxidative burst that is visible within 18 h after pathogen inoculation , which could be potentiated by SA as part of a positive feedback loop between this hormone and reactive oxygen species . On the other hand, RR fruit from NahG and wild-type plants were equally susceptible to B. cinerea and no necrotic response was evident with either genotype . These results suggest that unripe MG fruit are capable of promoting SA-mediated responses, possibly independently from those influenced by NPR1 ,and thereby, may prime fruit for resistance without favoring susceptibility.The increase in expression of JA biosynthetic and the subsequent accumulation of JA occurs locally as a consequence of pathogen, insect or physical damage to plant tissues . Up-regulation of three tomato homologs encoding JA biosynthetic enzymes, allene oxide synthase , 12-oxo-cis-10,15-phytodienoic acid reductase 3 , and 3-oxo-2– cyclopentane-1-octanoic acid -8:CoA ligase was observed during infections of MG and RR fruit . The expression of the OPR3 homolog was confirmed in B. cinerea-infected fruit after 1 and 3 dpi . In addition, up-regulation of a JAR1 homolog is detected in RR fruit at 1 dpi , but at 3 dpi its expression is down-regulated in both MG and RR tissues . JAR1 is a GH3 acyl-adenylase that conjugates isoleucine to JA, activating the hormone and it is required to activate JA-related responses of Arabidopsis leaves against necrotrophic infection . In the microarray data, transcriptional changes in response to B. cinerea are only evident for homologs of two downstream JA-responsive factors and a member of the SCFCOI1 complex . Transcriptional reprogramming of important JA-signaling components was not evident during tomato fruit infection or during ripening , which may indicate that activation of JA-related defenses in fruit occurs via other signaling pathways. In contrast, when B. cinerea infects petunia flowers it was been reported that expression of COI1 is activated in the absence of ET signaling , which indicates that JA signaling pathways could be differentially activated as consequence of fungal infection depending on the plant tissue and the presence/absence of endogenous ET levels. Both JA and ET synergistically activate the expression of a large set of defense genes through the transcription factors, ERF1 and ORA59 . These shared JA- and ET-regulated responses are preferentially triggered when ET is present, while responses unique to JA are induced mostly in the absence of ET . The antagonism is dependent on NPR1 and influenced by the hormone concentration and the timing of the SA/JA signal initiation . This interplay between SA and JA might reduce fitness costs from the unnecessary deployment of defenses and could serve as a regulatory mechanism allowing plants to adjust their defense strategies in response to the pathogen’s lifestyle .