In MG fruit, all three fungi were able to grow on the surface, but none of the pathogens was able to cause rot. In contrast, ripe tomato fruit represented a compatible system for infections as all three fungi induced lesions that spread rapidly. This contrasting ability to cause disease in fruit has been previously reported for a variety of fungal pathogens, particularly those displaying necrotrophic behavior . The tomato gene SlWRKY33 has been shown in leaves to be expressed in response to B. cinerea inoculation, and deletion of this gene leads to increased susceptibility, indicating its role in defense response . We demonstrated that, as in leaves, B. cinerea is capable of inducing SlWRKY33 in MG and RR fruit. Furthermore, F. acuminatum also induced SlWRKY33 in MG and RR fruit, and R. stolonifer did so substantially in RR fruit. These findings indicate that all three pathogens triggered disease responses in the host and that the strength of the response was reflective on the success of the infection process. During interactions with tomato fruit, B. cinerea, F. acuminatum, and R. stolonifer employed a variety of pathogenicity and survival strategies that involved redox processes, drainage collection pot carbohydrate catabolism, and proteolysis. Moreover, the degree to which particular strategies were used varied according to the ripening stage of the fruit, as certain processes were emphasized in either MG or RR fruit . These observations suggest that the fungi can sense the physiological environment of the fruit and react accordingly with suitable infection, growth, or quiescence strategies.

Though these fungi are incapable of causing disease symptoms in MG tomato fruit, this study demonstrates that they do make attempts to either establish infections or create a suitable environment in fruit for fungal growth and do not merely die on the host tissues. However, when the conditions in fruit are highly unsuitable , the infection strategy of the fungal pathogen is often insufficient to cause successful infections. In many cases, when fungal pathogens encounter incompatible conditions, like in unripe fruit, they enter a quiescent phase with limited growth and activity . During ripening, the physicochemical properties of the fruit tissues change, resulting in compatible conditions for the fruit-pathogen interaction and the reactivation of quiescent pathogens . In this context, it would also be interesting to investigate the strategies employed by the three fungi during inoculations of other plant organs such as leaves. Our initial tests, however, indicated that both F. acuminatum and R. stolonifer are incapable of infecting tomato leaves even when leaves were senescing. This observation may suggest that the isolates of these two fungi are exclusive fruit pathogens and lack the molecular toolset to grow on leaves. The redox environment of the plant-pathogen interface influences the outcome of the interaction. Upon pathogen detection, ROS are rapidly produced by the host, triggering a downstream signaling of various defense responses . The enzymatic agents of this oxidative burst are respiratory burst oxidative homologs , which generate superoxide O − 2 in the apoplast . This oxidative burst has been previously reported in incompatible tomato-Botrytis interactions , including MG fruit, in which the appearance of a necrotic ring is associated with resistance to B. cinerea . However, necrotrophic pathogens can exploit this ROS response by overwhelming the host with their own ROS production . In leaves of French bean , B. cinerea has been shown to produce ROS as virulence factors by activating the NADPH oxidases BcnoxA and BcnoxB , coupled with the regulatory protein BcnoxR .

Although we did not detect strong upregulation of these genes during inoculation of fruit, other ROS producing systems, including laccases and glucose oxidases were upregulated during inoculations of tomato fruit. In F. acuminatum, a BcnoxA homolog FacuDN4838c0g1i1 and BcnoxB homolog FacuDN3221c0g1i1 were induced in specific treatments. A BLAST search did not reveal anyhomologs of BcnoxA or BcnoxB in R. stolonifer, nor were any homologs of Bclcc8 or BcGOD1 detected in either F. acuminatum or R. stolonifer. In addition to ROS generation machinery, fungal pathogens must protect themselves against the oxidative stress of the infection site. Methods of ROS scavenging in phytopathogenic fungi include enzymatic and non-enzymatic mechanisms . SODs catalyze the conversion of O− 2 produced by RBOHs into the less reactive hydrogen peroxide . B. cinerea mutants lacking the BcSOD1 gene have been shown to have reduced virulence on tomato leaves . In tomato fruit, BcSOD1 is upregulated for both MG and RR ripening stages, which suggests it is also a critical gene for fruit colonization. H2O2 can be converted to water by either catalases or peroxidases such as GPXs or PRXs. All three pathogens demonstrated upregulation of specific mechanisms of catabolizing H2O2, but only F. acuminatum showed enrichment of genes involved in the H2O2 catabolic process. The usage of these H2O2 catabolizing systems varied between the pathogens. While B. cinerea utilized catalases in MG fruit at 1 dpi, F. acuminatum and R. stolonifer produced more catalases and peroxidases in RR fruit at 1 dpi. In each pathogen, multiple genes involved in protein degradation were found to be upregulated during fruit inoculations. The strong enrichment of proteolysis-related genes may indicate that protein degradation is important for pathogenicity of F. acuminatum and R. stolonifer but not B. cinerea. Some pathogen-derived proteases, such as Sep1 and Mep1 in Fusarium oxysporum, are known to serve as suppressors of host-immune response in plant-pathogen interactions .

Even though their specific roles in pathogenesis are not fully characterized, several aspartic proteinases in B. cinerea have been described . Three of the B. cinerea aspartic proteinases that we found to be induced in tomato fruit were also found to be upregulated during infection of grape berries . Aspartic proteinases were also found to be among the upregulated proteinases in F. acuminatum and R. stolonifer , though all three pathogens appeared to utilize a diverse suite of proteinases of different families. Especially prominent in F. acuminatum and R. stolonifer were proteins with similarity to subtilisin-like proteases. This family of enzymes is mostly associated with plants and particularly plant defense, but subtilisin-like proteases involved in pathogenicity have been described for fungi as well . Since these inhibitors possess sequence similarity to the proteases themselves, the enzymes identified in F. acuminatum and R. stolonifer may be inhibitors, proteases, or a mixture of both. Additionally, proteases can help with host tissue decomposition by breaking down cell wall structural proteins or can serve in degradation of proteins to provide a source of nutrition for fungal growth . For example, the saprotrophic fungal species Verticillium albo-atrum and V. dahliae were described to secrete proteases to break down structural proteins that stabilize the plant cell walls . High proteolytic activity resulting in the degradation of proteins into free amino acids was also reported during fermentation of tempeh by several Rhizopus species . Botrytis cinerea, F. acuminatum, and R. stolonifer also make use of a variety of CAZymes during interactions with the host. Several CAZyme families are involved in the breakdown of physical barriers present in the host tissues, namely the various cell wall components , cell wall reinforcements , and the waxy fruit cuticle. Many of these enzymes, such as polygalacturonases, pectin methylesterases, pectate lyases, and endo-β-1,4-glucanases, square plastic pot mirror the activities of host enzymes active during the ripening-related softening of the fruit . Others, such as cellulases, cutinases, and lipases, degrade components that are not typically degraded during ripening. Production of cellulases is also coupled with enzymes involved in degradation of cellobiose, the disaccharide product of cellulose breakdown. Both B. cinerea and F. acuminatum appear to focus on production of these latter CAZyme families in MG fruit more than in RR fruit. This may be due to the greater strength and integrity of the cell wall in MG fruit, which requires the fungus to mount a larger attack on the physical barriers in order to penetrate into the cells. Degradation of pectin is a hallmark feature of B. cinerea infection of plant tissues .

The principal enzymes responsible for this process are polygalacturonases , pectin methylesterases , and pectate lyases . Both PGs and PLs cleave the α- 1,4-linkages in the homogalacturonan backbone of pectins. PMEs catalyze the removal of methylester groups on the C6 carbons of galacturonan, which allows for further degradation by PGs. Although overexpression of PME inhibitors in Arabidopsis leaves has been shown to increase resistance to B. cinerea , mutations in Bcpme1 and Bcpme2 do not appear to affect virulence in tomato leaves . In B. cinerea, all three classes of enzymes appear to be highly expressed in MG fruit but not as prominently in RR fruit. Not only do the GH28, PL1-7, and PL3-2 families constitute a greater fraction of upregulated CAZymes in MG fruit, but for PGs, PLs, and PMEs that are commonly upregulated in MG and RR fruit, upregulation is consistently greater in MG fruit over RR fruit. Additionally, although no F. acuminatum PGs were detected in MG, the two upregulated PMEs, FacuDN5818c0g1i1 and FacuDN10179c0g1i1, were only active in MG fruit. Moreover, PL1-7 and PL3-2 genes were strongly expressed in MG fruit, with one PL3- 2 gene, FacuDN8473c0g1i1, showing a log2FC of 10.29 at 1 dpi, the highest of any plant CWDE in this treatment. Only one R. stolonifer PG, RstoDN2036c0g1i1, was detected in MG fruit. However, given that this single R. stolonifer PG was one of only two CAZymes found in 1 dpi MG fruit, it is reasonable to believe PG activity in R. stolonifer isbeing underestimated due to low sequence coverage of fungal transcripts in this treatment. The absence of upregulation of any R. stolonifer pectate lyases in any fruit further underscores this point. Given the prominence of pectin degradation in B. cinerea and F. acuminatum, a more targeted analysis of R. stolonifer pectin degradation, especially in MG fruit, is warranted. Degradation of the host cell wall in MG fruit by pathogen enzymes may accelerate ripening and in turn facilitate a more favorable environment for colonization. Pectinderived oligosaccharides have been shown to induce ethylene production in tomato fruit , which further upregulates expression of host CWDEs, including PG. B. cinerea can synthesize its own ethylene via the α-keto-γ-methylthiobutyric acid pathway , though it is still unknown whether the pathogen produces ethylene during interactions with the fruit. Ethylene production during plant infection has also been reported via the KMBA pathway for species of Fusarium , but not, to our knowledge, for R. stolonifer. However, the specific genes involved in the KMBA pathway in B. cinerea or Fusarium spp. have yet to be elucidated. As colonization proceeds, sugar substrates become available due to degradation of cell wall polysaccharides as well as increased access to stored sugars in the fruit. As a consequence, fungi actively infecting RR tomato fruit induced enzymes that metabolize simple sugars. Sugar metabolism is accompanied by expression of CAZyme families involved in the production and modification of chitin, the structural component of fungal cell walls. Chitin production is known to be a hallmark of growth for fungal pathogens . Interestingly, chitin production and modification appear to be prominent not only in RR fruit for each pathogen, but also in MG fruit inoculated with F. acuminatum, where a much greater amount of mycelia growth was observed compared to the other two pathogens. The equal representation of CE4 enzymes in MG and RR fruit inoculated with F. acuminatum is reflective of the ability of this fungus of producing hyphae at either fruit ripening stage. The abundance of polysaccharide-building glycosyltransferases in RR infections with R. stolonifer is also likely connected to the abundant mycelial growth. Other CAZyme families represent more specialized roles in the infection process. Production of enzymes in the AA7 family may be related to the production of polyketide toxins in B. cinerea and R. stolonifer. B. cinerea is known to produce botcinic acid, a polyketide mycotoxin, during infection . However, the AA7 genes detected to be upregulated in fruit infection here are not known members of the botcinic acid pathway, suggesting that B. cinerea may produce additional uncharacterized polyketide mycotoxins during fruit infection. Even though upregulated F. acuminatum genes involved in toxin production are not annotated as members of the AA7 family, fumonisins are products of polyketide metabolism . The observed upregulation of fumonisin biosynthesis related genes indicates that F. acuminatum also produces polyketide mycotoxins during infection of unripe and ripe tomato fruit.