In immature fruit, enriched pathways were more evident at or after 24 hpi. In contrast, multiple pathways were enriched in mature fruit, as shown by early time points, which suggests an overall activation of stress responses associated with the biotic challenge and tissue breakdown. These time-dependent responses to M. laxa were also evident when quantifying the number of DEGs for enriched categories related to plant defense , which confirmed that immature fruit had the highest gene expression induction at 24 hpi, and that mature fruit had a larger number of genes induced than immature fruit as early as 6 hpi. DEGs related to the plant–pathogen interaction pathway were largely absent from the immature fruit response, with the exception of 24 hpi, but were quite abundant in the mature fruit response starting at 14 hpi . Hormone signaling was enriched early in fruit at both developmental stages, though it appeared to become less relevant in immature fruit at 48 hpi. Cysteine and methionine metabolism and α-linolenic acid metabolism pathways, associated with ethylene biosynthesis and jasmonic acid biosynthesis, respectively, were enriched in both immature and mature fruit, though more prominently in the latter. Pathways related to the biosynthesis of terpenoids were also found to be enriched at early time points in immature and mature fruit , but their enrichment was higher in immature than mature tissue. Other pathways that appeared to be relevant for nectarine responses against M. laxa included the phenylpropanoid and glutathione metabolism,dutch buckets which were highly induced in the mature fruit, likely utilized as antioxidants.
Given the enrichment of genes involved in plant hormone signaling transduction during early infection and the activation of methionine and α-linolenic metabolism in both fruit tissues across time, a targeted analysis of ET and JA pathways was conducted. The transcriptional activation of JA biosynthesis was evident in immature and mature fruit, with special emphasis in the induction of multiple genes encoding the initial biosynthetic steps , from lipoxygenase to 12-oxophytodienoic acid reductase . Later steps of the biosynthesis pathway were only moderately activated in both tissues. In mature tissues at 48 hpi, a down regulation of the JA-amino synthetase gene was observed, involved in the production of the active form of JA, and of the homolog of the JA receptor coronatine-insensitive protein 1 . Two out of the five paralogs of the signaling repressor JA ZIM domain appeared to be activated in immature and mature tissues at multiple time points. The three paralogs encoding the transcriptional activator of JA responses, MYC2, were strongly induced in mature fruit after 14 hpi and upregulated in immature fruit only at 14 hpi and 24 hpi. In fact, the MYC2 gene expression level of the third paralog was significantly higher in inoculated immature than mature tissue, but then, its expression was significantly higher in mature than immature tissue at both 24 and 48 hpi . The steps committed to ET biosynthesis catalyzed by the 1-aminocyclopropane-1-carboxylate synthase and the 1-aminocyclopropane- 1-carboxylate oxidase genes were highly induced in response to M. laxa inoculations, particularly in mature fruit .
Ethylene signal transduction elements showed only moderate changes in gene expression in response to the pathogen. Interestingly, although the negative regulator EBF1/2 was down regulated at 14 and 48 hpi in both tissues, it was highly upregulated in immature tissue at 6 and 24 hpi. However, all three paralogs of the ET response factor 1/2 , which control multiple ET responses and are a point of signal integration for JA and ET signal transduction, were highly upregulated in both tissues. The ERF1/2 gene expression level of the second paralog was significantly higher in mature inoculated than immature inoculated fruit at 14 hpi . In addition, the ET produced by M. laxa-inoculated and control fruit was measured to complement the transcriptional data . Control nectarines followed the ET pattern of a climacteric fruit; low and steady levels of ET in immature fruit and high and significantly increased levels in mature fruit until ripening. However, in inoculated immature fruit, ET production significantly peaked at 24 hpi, corresponding to the peak of transcriptional responses in this tissue, before returning to levels equivalent to the control fruit. In inoculated mature fruit, the ET production was significantly lower than control fruit at 6 hpi, but then significantly increased. These results suggest that nectarine was performing a tightly regulated response of ET.To determine which fungal genes and functions are biologically relevant during M. laxa interactions with nectarine, we performed a functional analysis of the pathogen transcriptome. First, a total of 9581 transcripts were denovo annotated for multiple functional categories, including carbohydrate-active enzymes , fungal peroxidases , genes involved in pathogen–host interactions , membrane transport proteins , and proteins with signal peptides , among others . Then, an enrichment analysis of these large functional categories in the upregulated DEGs across infection was performed to obtain a general picture of specific gene categories induced by the pathogen in immature and mature fruit .
In immature fruit, these large categories were enriched in M. laxa upregulated DEGs at least at one time point when compared to 6 hpi. Particularly at 24 hpi, a significant abundance of CAZymes and PHI genes was observed. Fungal peroxidases were only significantly enriched in immature fruit at 48 hpi. In contrast, enrichment of CAZymes and fungal peroxidases was not observed at any time point in mature tissues. Genes in involved in pathogen–host interactions and membrane transport remained enriched at relatively even levels from 14 to 48 hpi in mature fruit. We identified GO terms related to pathogenicity, virulence, and fungal growth among the upregulated DEGs for each host developmental stage . Among this subset of biologically relevant GO terms, threefold more upregulated DEGs were detected when M. laxa was inoculated in mature fruit compared to immature fruit. Particularly, the number of M. laxa upregulated DEGs in immature tissue increased progressively until 24 hpi and then decreased slightly at 48 hpi, whereas in mature tissue, the upregulated DEGs increased along with infection time. Notably, these gene expression patterns resembled the transcriptional response of the host for each developmental stage . In both stages, M. laxa induced a high number of DEGs related to oxidative–reduction processes and transmembrane transport, although genesinvolved in protein translation and proteolysis were only abundantly expressed in mature tissue. However, genes involved in response to oxidative stress were mainly expressed in immature at 48 hpi, together with the enrichment of fungal peroxidases at this time point . Lastly, the enrichments of Pfam domains were also carried out using the M. laxa upregulated DEGs . In agreement with previous results, Pfam categories were mainly enriched at 24 hpi in immature fruit, with the exception of proteins containing the fungal pathogenesis-related CFEM domain, which were uniquely enriched earlier at 14 hpi. In addition, Pfam domains related to fungal membrane transport were largely prominent in immature fruit, especially at 24 hpi, where up to 53 genes were induced. Less significantly enriched, fungal glycosyl hydrolases, dehydrogenases , and catalases were found at 48 hpi in immature tissues. The number of enriched Pfam domains among M. laxa upregulated DEGs in mature fruit, such as those related to transcription and translation , increased throughout disease progression . However, other relevant domains, such as some related to proteolysis activity , uniquely peaked at 14 hpi. Notably, upregulated DEGs annotated as ribosomal proteins and transcriptional factors involved in growth and cell cycle control were prevalent throughout infection of mature fruit. Later,grow bucket infection time points exhibited enrichments of protein domains belonging to membrane transport and redox functions .To identify potential target genes for the control of M. laxa, a closer examination was conducted of the most highly M. laxa upregulated DEGs from all time points and tissue comparisons . The top five M. laxa-induced DEGs in immature and mature fruit were unique between the tissue types, reinforcing the evidence that the pathogen displays a different behavior according to the developmental stage of the host. Strongly induced DEGs at 14 hpi unique to early infections of immature fruit included fungal phosphate transporters, phospholipases, and oxidoreductases. A member of the glycosidase hydrolase family 31 was highly expressed at 24 hpi in immature fruit, alongside a transmembrane fructose transporter and histidine phosphatase . The highest induced DEGs in immature fruit were detected at 48 hpi and corresponded to an oxido reductase gene , a homolog of the alcohol oxidase from Cladosporium fulvum, and the same transmembrane fructose transporter already found at 24 hpi. Interestingly, M. laxa DEGs with fungal peroxidase annotations, a catalase and a haloperoxidase , were only detected at 48 hpi in immature fruit. In mature fruit, a single protease gene was the highest upregulated M. laxa DEG at all time points. Two polygalacturonases were among the largest induced DEGs during infections of mature fruit; Monilinia_000560 was highly upregulated at 14 hpi, whereas Monilinia_041700 was highly expressed at 24 and 48 hpi.
Another CAZyme was also highly enriched at 14 and 24 hpi. In mature tissue, transporters and hormonerelated genes were among the highest expressed DEGs. An amino acid transporter was significantly expressed at 14 hpi, while a tryptophan 2- monooxygenase was induced at 48 hpi, known to be involved in virulence in another pathosystem. Altogether, these results suggest that targeting of specific genes involved in response to oxidative stress, nutrient transport, and carbohydrate catabolism may reduce quiescent infections, while specific proteolytic genes and additional CAZymes may help inhibit or reduce the severity of disease in susceptible fruit.The first line of plant defense that M. laxa has to overcome is the constitutive physical and chemical barriers present in the fruit surface. The developmental process from immature to mature fruit is characterized by physical and chemical changes in fruit firmness, leading to softening at the onset of ripening. In fact, the flesh firmness of immature fruit was higher than the mature fruit . Monilinia laxa appeared to produce more CWDE in immature fruit, which suggests that the pathogen could be trying harder to overcome the host cell walls in these tissues. Nevertheless, the immature tissue had no visible disease symptoms. Other alterations occurring during fruit development include changes in plant cuticle, sugar accumulation, volatile compounds, and secondary metabolites synthesis, which have been reviewed as promoting susceptibility to pathogens in ripening fruit. Hence, higher soluble solids content and lower titratable acidity on mature fruit could favor pathogen colonization. Plant–pathogen interactions take place when pathogen associated molecular patterns are recognized by the plant’s pattern recognition receptors, which ultimately triggers a defense response known as PAMP triggered immunity. The chitin elicitor receptor kinase 1 was upregulated in the mature tissue at 14 hpi. Also, the expression levels of the transcriptional activator PTI5 were up to 2.5-fold and 5-fold higher in mature fruit when compared to immature fruit, at 24 and 48 hpi, respectively. PTI responses can be suppressed by effector proteins secreted by the pathogen, which in turn, will elicit effector-triggered immunity. In our pathosystem, proteins with the CFEM domain and signal peptides were enriched in the early infection stage on immature tissue. Among the annotated genes with the CFEM domain, the Monilinia_077410 is a homolog of BcCFEM1 from B. cinerea, an effector shared by many Botrytis spp.and described to be important for its virulence. These results suggest that M. laxa may secrete some type of effector proteins in immature fruit. Once the host–pathogen interaction began, both pathogen and host triggered their own transcriptional reprograming. In mature tissue, both nectarine and M. laxa abruptly changed their gene expression profile at 14 hpi, coinciding with the ability of the pathogen to grow and macerate the fruit tissues within 14 h. From 14 hpi onwards, the pathogen started to penetrate and switched toward an aggressive necrotrophic phase, which was retained at later infection times. Functions related to transmembrane transport, oxidation-reduction process, and translation were among the most abundant activities in mature fruit, denoting the growth and spread of the pathogen. In contrast, the number of nectarine and M. laxa DEGs in immature fruit remained somewhat steady through infection time, even when fungal biomass peaked at 24 hpi. Overall, these findings suggest that inoculated mature nectarines displayed an earlier and broader response to M. laxa than immature ones, likely due to the faster pathogen growth and virulence mechanisms activation in these tissues. Both PTI and ETI are able to induce the host hormone signaling transduction pathway, which was found to be enriched, starting at 6 hpi in both tissues.