C. sativa is a European woody tree species commonly used across the globe in the food and timber industries. This chestnut species is susceptible to the two major pathogens, P. cinnamomi and C. parasitica [10,36]. In contrast, the Asian chestnut species C. crenata and C. mollissima exhibit higher tolerance to P. cinnamomi and C. parasitica [6]. Achieving tolerance or resistance to pathogens is the major aim of rootstock breeding. Blight-resistant trees were obtained through backcross breeding of introgression genes from Asian species into American chestnut trees. [37]. However, this approach, although successful in developing blight-resistant American chestnut selections has been slowed by a lack of genetic tools. In Europe, ink disease tolerant hybrids were obtained through interspecific crosses between C. sativa and C. crenata, although the nut quality produced by these hybrids is below current market standards [38,39].

It has long been recognized that a deep understanding of a pathogen’s biology, host–pathogen interactions, and the resistance mechanisms are fundamental to improving breeding programs. Genomic and transcriptomic analyses have provided the first genetic insights into mechanisms underlying susceptible and resistant chestnut species responses to P. cinnamomi and C. parasitica [37,38,40,41,42]. Santos et al. [40] reported the upregulation of a set of candidate genes (e.g., Cast_Gnk2-like and Calcium-dependent protein kinase) after P. cinnamomi infection, which may trigger HR-like cell death in C. crenata cells. A significant number of genes involved in the defense against chestnut blight were identified [37].

Traditionally, the introduction of resistance gene analogues into plants was the most promising approach to facilitate the acquisition of resistance. However, it did not prove to be durable enough because the widespread use of R genes caused the selection of pathogens capable of overcoming them [24]. Susceptibility (S) genes can be interesting candidates to be used in target breeding programs [22,23,24]. Furthermore, on the basis of previous studies, it was highlighted that the disabling of susceptibility genes may facilitate durable resistance since the pathogen needs to gain a new function to replace the lost host factor it was exploiting [43].

In woody species, the investigation of S-genes has been performed only for MLO genes in rubber trees [32], poplar trees [33], apple trees, and grapevines [34,44]. Due to the absence of a C. sativa genome, highly similar S-genes were selected using the C. mollissima v 1.1 genome. Based on the blastn survey, four loci with high similarity were identified in the C. mollissima genome and attributed to different subclasses of S-genes [31,45,46,47] due to the presence of specific domains: mlo1, dmr6, dnd1, and pmr4 (Figure 1, Table 1). As previously observed [31], in the phylogenetic trees, monocot proteins formed a separate clade with respect to those of dicotyledonous species, supporting the hypothesis that an independent evolution occurred for these genes (Figure 2). Quantitative PCR analysis has been carried out to identify the differential expression of candidate S-genes in response to P. cinnamomi and C. parasitica in the stems of a susceptible species, C. sativa, and of a tolerant one, C. crenata. Lesion analysis and DNA quantification of the pathogen (Figure 4 and Figure 6) confirmed the higher tolerance level of C. crenata in response to both P. cinnamomi and C. parasitica infection. Our qPCR results highlighted the main upregulation of pmr4 and dmr6 in response to infection by both P. cinnamomi and C. parasitica. As expected, a greater increase in the transcription of these susceptibility genes was observed in the susceptible species C. sativa. Remarkably, pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa; in the tolerant C. crenata, significant upregulation was observed (Figure 5 and Figure 7). Pmr4 codifies for a callose synthase, which is necessary to create a physical barrier to avoid pathogen penetration and is also implicated in plant-triggered immunity suppression. Pmr4 is thus not only involved in the synthesis of callose, but it also acts as a negative regulator of the salicylic acid pathway [28].

Huibers et al. [48] demonstrated that resistance due to the silencing of Pmr4 is associated with salicylic acid (SA) accumulation rather than with the callose deposition absence. Salicylic acid signaling plays a key role protecting against biotrophic pathogens through the establishment of a hypersensitive response (HR). Saiz-Fernandez et al. [49] revealed the increment of SA levels in P. cinnamomi inoculated stems, indicating that P. cinnamomi activates a defense response similar to that triggered by biotrophic pathogens. Inoculation with both virulent and hypovirulent strains of C. parasitica led to SA accumulation in European chestnut plantlets that were grown in vitro [50]. Transcriptome analyses carried out in both C. dentata and C. mollissima highlighted activation of salicylic-acid-related genes in canker tissues [37].

In chestnut trees, callose deposition around P. cinnamomi hyphae was detected early in the infection process; however, it does not seem to play a key role in the associated interactions since the pathogen can reach the vascular cylinder in both susceptible and resistant plant genotypes [51]. This result was validated by transcriptomes analyses of C. sativa and C. crenata, in which no overexpression of Callose synthases after P. cinnamomi infection was observed [38].

Based on our results and the literature, we can hypothesize that callose accumulation due to the pmr4 upregulation in inoculated C. sativa lines may not be responsible for controlling P. cinnamomi colonization. We suggest that the upregulation of pmr4 could lead to a negative regulation of the SA pathway that in turn provokes the susceptibility of C. sativa to both P. cinnamomi and C. parasitica. A clear link with SA pathway has emerged even with the other chestnut gene candidate dmr6 (downy mildew resistance 6). The mutation of Arabidopsis dmr6 gene, associated with salicylic acid (SA) homeostasis [31], results in the generation of plants that are resistant to bacteria and oomycetes. Dmr6 is involved in the conversion of salicylic acid (SA) to 2,3-dihydroxybenzoic acid (2,3-DHBA) and negatively regulates the expression of defense genes (PR-1, PR-2, and PR-5) [30].

The expression trend of the Dmr6 gene in response to P. cinnamomi infection turned out to be similar to the profile of pmr4. Indeed, dmr6 was strongly expressed at early infection phases of P. cinnamomi in C. sativa; in C. crenata no significant upregulation was detected (Figure 5). No upregulation of dmr6 in response to C. parasitica was highlighted in both plant species (Figure 7). We can thus hypothesize that dmr6 upregulation observed in C. sativa could negatively regulate defense gene expression, leading to susceptibility to P. cinnamomi.

Plants produce a variety of hydrolytic defense enzymes against pathogens, including chitinases, proteases, and also glucanases [52]. The genes coding for several pathogenesis-related (PR) proteins, Acidic 26 kDa endochitinase gene (chi3) and Glucan endo-1,3-beta-glucosidase B gene (gluB), were selected in our analysis because they are considered as responsive to SA-dependent signaling [53,54]. Chi3 and gluB are enzymes that cause the lysis of hyphae of various pathogens, resulting in growth inhibition [55,56,57].

In both C. sativa and C. crenata plants inoculated with C. parasitica, chi3 and gluB were significantly upregulated. The transcription of chi3 was higher in C. crenata than in C. sativa, presumably as a consequence of the improved defense mechanism against C. parasitica. Our results are in agreement with Shain et al. [58], who demonstrated the involvement of b-1,3-glucanase and chitinase in chestnut species affected by C. parasitica. Studies on the role of chitinase in blight infection mostly involved C. sativa as a model system [50,59]. In both C. dentata and C. mollissima, transcripts of several compounds expressing chitinase accumulated more in canker tissues than healthy stem tissues [37]. In order to obtain chestnut plants with potentially increased resistance/tolerance to chestnut blight, the endogenous Ch3gene encoding a chitinase-like protein was over-expressed in the European chestnut through Agrobacterium-mediated transformation [60].

The emergent CRISPR/Cas9 technology is expected to play a key role in future crop breeding as it allows highly efficient gene editing and generates genetic changes indistinguishable from those arising spontaneously in nature or through conventional breeding [61]. Several examples of edited plants resistant to fungal pathogens have been described [62,63]. For example, genome editing was successfully applied to knock out mlo S-genes, leading to Powdery mildew (PM) resistance [44,64,65,66]. Pmr4 and dmr6 loss-of-function through CRISPR/Cas reduced the susceptibility to PM in tomato plants [28,67]. In our laboratory, we are setting up a CRISPR/Cas9 transformation protocol in Castanea sativa. Our future goal will be to perform the functional characterization using the CRISPR/Cas9 approach of the two candidate genes (dmr6 and pmr4), while checking if the two genes may also play a role in the interaction between C. sativa and the emergent nut rot and canker agent Gnomoniopsis castaneae [68].