Using the fungal ortholog dataset of the Benchmarking Universal Single-Copy Orthologs tool , we determined that our assemblies presented high completeness, with 88.2 and 90.3% of F. acuminatum and R. stolonifer matchesbeing complete, respectively. Our F. acuminatum transcriptome contained 20,117 unique transcripts, while our R. stolonifer transcriptome contained 19,754 . We then used homology-based annotation to obtain information on gene functions for each of the transcriptomes, including the B. cinerea B05.10 ASM83294v1 . We annotated transcripts based on nine separate functional classifications, including GO , Pfam domains , Pathogen-Host Interaction , membrane transporters , Carbohydrate-Active Enzymes , and fungal peroxidases . Each type of functional annotation was represented by a similar percentage of annotated transcripts across all pathogens . The specialized enzyme classifications of peroxidases and CAZymes made up a relatively small fraction of the annotated transcripts, whereas general functional classifications such as GO, Pfam, and KEGG descriptions were available for at least 70% of the annotated transcripts for all pathogens. Annotations for all three transcriptomes can be found in Supplementary Table S3. Although the F. acuminatum and R. stolonifer transcriptomes are preliminary and may require further curation and validation, pot with drainage holes we consider that they are a valuable resource to perform gene expression analyses and to shed light on the infection strategies utilized by these fungi.

First, we performed principal component analysis to determine if the fungal-inoculated and in vitro samples could be discerned based on the expression of the fungal transcripts. The PCAs revealed that all samples clustered by treatment . In most cases, the first component clearly differentiated the MG fruit from the RR fruit inoculations and the in vitro samples. Then, we determined DEGs between inoculations of MG or RR fruit and in vitro cultures for each pathogen. Across all comparisons, we detected 6,488 B. cinerea DEGs , 6,154 F. acuminatum DEGs , and 8,777 R. stolonifer DEGs . The number of DEGs for R. stolonifer were mainly identified in the RR fruitcomparisons, as the low amount of fungal biomass in MG fruit samples did not allow for an in-depth sequencing coverage of the fungal transcripts. To confirm the accuracy of the DEG analysis, we selected a subset of genes for each pathogen to validate their expression using a qRT-PCR approach . Our results confirmed that the gene expression values were consistent, showing significant Pearson correlation coefficients and between the RNA-seq and the qPCR expression data . We further evaluated the fungal DEGs based on whether they were commonly or uniquely expressed under specific treatments, which can provide insight on particular sets of genes that are relevant during incompatible or compatible interactions . For each pathogen, genes uniquely upregulated in RR fruit constituted a sizable fraction of upregulated genes . This result may be influenced by the fact that RR fruit samples had more coverage of fungal transcripts in the RNA-seq experiment than MG fruit samples, which is a technical limitation of this type of study. Nevertheless, the comparisons of common and unique DEGs among treatments for each of the pathogens support the results of the PCAs, indicating that these fungi display a specific behavior in each of the fruit stages at early and late time points after inoculation.

We also identified upregulated DEGs shared across categories that are likely to represent core pathogenicity factors during fruit infections.To gain insight into key biological processes that are relevant during compatible or incompatible fruit infections, we performed GO enrichment analyses of the upregulated DEGs in all combinations of ripening stage and dpi for each pathogen . We mainly focused on GO terms of the “biological process” class that were significantly enriched and appeared to be involved in pathogenesis or fungal growth in the host tissues . Upregulated DEGs from all comparisons, except for R. stolonifer MG inoculations, were enriched in oxidationreduction processes . A closer inspection of these DEGs revealed functions that are likely to be involved with pathogenicity, such as catabolism of ROS [e.g., superoxide dismutases , catalases , peroxidases] and breakdown of cell wall molecules such as cellobiose and lignin . In B. cinerea, the SOD BcSOD1 was induced in both MG and RR fruit at 1 and 3 dpi. Additionally, BcSOD3 was upregulated only in MG fruit at 1 dpi, and BcSOD2 is upregulated only at 1 dpi in MG and RR fruit. Although two potential SODs, FacuDN9613c0g1i1 and FacuDN4275c0g1i2, were employed by F. acuminatum in all treatments except 1 dpi MG, none of the seven putative SODs identified in R. stolonifer were upregulated in any of the treatments. To further identify enzymatic scavengers of hydrogen peroxide , we examined the upregulated DEGs of each pathogen which showed significant similarity to members of the Fungal Peroxidase Database. This analysis revealed differences both in the classes of enzymes used in each pathogen and the treatments in which they were used. For example, in B. cinerea, only two known catalases, BcCAT2 and BcCAT4 , were found to be upregulated during tomato fruit interaction.

Both of these were only active in MG fruit. In contrast, F. acuminatum exhibited very strong induction of two predicted CATs, FacuDN12367c0g1i1 and FacuDN13048c0g1i1, at 1 dpi in RR fruit but not in MG fruit, although a handful of CATs and catalase-peroxidases were upregulated less strongly across both MG and RR fruit. In all F. acuminatum-inoculated samples, there was also an enrichment of DEGs involved in hydrogen peroxide catabolism , further highlighting the importance of fungal responses to oxidative stress during fruit colonization. In R. stolonifer, peroxidases were only upregulated at 1 dpi in RR fruit and included two 2-cysteine peroxiredoxins , one cytochrome C peroxidase, and one glutathione peroxidase . Additionally, in all B. cinerea-inoculated samples, DEGs annotated with the oxidation-reduction process GO term included enzymes in the biosynthetic pathways for the phytotoxins botrydial and botcinic acid. Eight of these genes were strongly upregulated in all four treatments, indicating that B. cinerea may produce these toxins regardless of the ripening stage of the fruit. F. acuminatum genes annotated with this GO term included enzymes involved in the biosynthesis of the toxin fumonisin. Several of these genes showed significant upregulation in infections of MG fruit at 1 dpi or RR fruit at both time points. Fungal proteolysis-related genes were found to be enriched during MG and RR inoculations with F. acuminatum as well as RR inoculations with R. stolonifer at 3 dpi. Though not enriched, several genes with this GO term were also found to be expressed during fruit inoculation by B. cinerea, mostly in RR fruit. Across all treatments, F. acuminatum was found to produce 28 genes with this GO term, while B. cinerea was found to produce 29, and R. stolonifer produced 44 in RR fruit alone . Seven members of the B. cinerea aspartic proteinase family wereupregulated in at least one of the fruit inoculations, though none were upregulated at 1 dpi in RR fruit. Thus, large pot with drainage fungal proteases are likely to be a strategy used by all three pathogens. Other GO terms served as a proxy for successful growth. Enrichments of genes involved in protein translation initiation , glycolytic process , and DNA replication initiation were found in compatible interactions with RR fruit. Notably, DEGs involved in glycolytic process were enriched in MG inoculations for F. acuminatum at 1 dpi, which is consistent with visual observations of mycelium growth on inoculated fruit. A similar pattern was observed for the chitin catabolic process term, which are involved in the continuous fungal cell wall remodeling during hyphal growth . Multiple GO terms relating to carbohydrate metabolism were found to be enriched across multiple fruit inoculation treatments. The corresponding genes included those involved in breakdown of the cell wall polysaccharides, metabolism of host sugar sources, and production of fungal polysaccharides. As both the cell wall properties and sugar biochemistry differ between MG and RR fruit, we hypothesized that the fungi employ different classes of CAZymes depending on the ripening stage as already demonstrated for B. cinerea . To test this, we examined the expression profiles of CAZyme families among the DEGs for each pathogen . CAZyme families involved in catabolism of cellulose, hemicellulose, pectin, and monosaccharides were detected, along with families with non-carbohydrate substrates and several responsible for polysaccharide biosynthesis. In B. cinerea and F. acuminatum inoculations, families involved in the degradation of cellulose and hemicellulose were more prominent during infections of MG fruit than RR fruit. Moreover, the CE5 family, which contains cutinases and acetylxylan esterases, was also especially utilized at 1 dpi in MG fruit. In B. cinerea, this family included the cutA gene previously shown to be expressed in tomato fruit infection .

MG infections also exhibited higher percentages of families involved in the degradation of cellobiose, a disaccharide of β-1,4-linked glucose molecules that results from the breakdown of cellulose and glucan-based hemicelluloses. A similar trend was found for pectin-degrading families, particularly polygalacturonases and pectate lyases , though the PL1-4 subfamily appeared to be prominent in RR infections as well. Enzymes involved in metabolism of simple sugars, most notably GH32 in F. acuminatum and AA3- 2 in B. cinerea, showed greater prominence in RR infections. Chitin and chitosan biosynthesis and processing families were also detected in B. cinerea and F. acuminatum. In B. cinerea, chitin synthases were generally equally expressed in all fruit inoculations, though chitin deacetylases , which produce chitosan, were only particularly prominent in RR infections at 3 dpi. In contrast, F. acuminatum produces multiple CE4 enzymes at 3 dpi in MG infections in addition to RR infections. Other CAZy families also seemed to be featured heavily in fruit-pathogen interactions. CE10 enzymes were especially prevalent in both B. cinerea and F. acuminatum infections. Members of the CE10 family include lipases, which catalyze the hydrolysis of fatty acids. The previously described B. cinerea gene lip1 was upregulated at both 1 and 3 dpi in MG fruit, but not RR fruit. Additionally, B. cinerea and F. acuminatum both produced multiple AA7 family enzymes in both MG and RR fruit. Many of these genes showed significant similarity to three genes of the PHI database: ZEB1 in F. graminearum, CTB5 from Cercospora nicotianae, and sol5 from Alternaria solani. Each of these PHI genes is involved in the biosynthesis of polyketide mycotoxins in those plant pathogens . Thus, these B. cinerea and F. acuminatum genes may be involved in similar roles. Detection of CAZymes during infection by R. stolonifer was only possible in RR fruit due to the low number of DEGs determined in MG fruit. However, sizable numbers of genes from families detected in B. cinerea and F. acuminatum infections were also discovered in R. stolonifer. These include xyloglucanases , cellobiose dehydrogenases , pectin methylesterases , and polygalacturonases . In addition, multiple enzymes involved in chitin/chitosan biosynthesis were prevalent in RR fruit inoculations, which is indicative of the particularly aggressive hyphal growth of R. stolonifer on these fruit. In RR fruit at 3 dpi, R. stolonifer also produced six enzymes of the AA1 family, which consist of laccases, ferroxidases, and multi-copper oxidases. Each of these enzymes showed significant similarity to FET3 enzymes from Colletotrichum graminicola in the PHI database and to genes of the TCDB class 2.A.108.1.4, the latter being iron transport multicopper oxidase FET5 precursors. This finding is also consistent with the enrichment of transmembrane transport genes during RR infection at 3 dpi for R. stolonifer.We inoculated fruit of the non-ripening tomato mutant to verify the effect of the ripening stage on the infection success of B. cinerea, F. acuminatum and R. stolonifer. Fruit from the nor mutant do not show ripening-associated processes, such as carotenoid and sugar accumulation or cell wall disassembly, and therefore resemble wild-type MG fruit even at a comparative RR-like stage. No hyphal growth of B. cinerea and R. stolonifer was apparent, whereas F. acuminatum formed visible mycelia especially at 3 dpi in MG and RR-like fruit. Like on wild-type MG fruit, all three fungi induced necrotic rings in nor fruit. When inoculated in RR-like fruit from nor, the three fungi displayed similar growth and morphology as in MG fruit from wild-type and nor, indicating that for compatible interactions to occur, tomato fruit needs to undergo certain ripening processes that facilitate fungal colonization and spread.Successful infections of B. cinerea, F. acuminatum and R. stolonifer in tomato fruit are dependent on the host developmental stage.