Gummy stem blight is one of the major diseases in cucumber (Liu et al., 2017; Zhang et al., 2017). Identifying candidate genes related to GSB resistance would enhance cucumber breeding and reduce crop losses due to this disease. However, the genetic mechanism of GSB resistance in cucumber is complicated and controversial. Furthermore, genetic sources of resistant are scarce in cucumber cultivars, and most germplasm used for GSB resistance in cucumber are wild cucumbers, including Cucumis hystrix and C. sativus var. hardwickii. The accession PI 183967 (C. sativus var. hardwickii) is the progenitor of cultivated cucumber, and carries valuable genes for resistance to GSB both in leaves at the seedling stage, and in stems at the adult stage (Liu et al., 2017; Zhang et al., 2017). Therefore, dissecting the genetic loci and determining mechanisms for GSB resistance in PI 183967, would allow for a translational breeding pipeline for disease resistance in cucumber cultivars.
Most studies on QTL mapping of GSB resistance are focused on seedling leaves, and few look at the stems of adult plants. In our previous study, we identified loci associated with GSB resistance on adult stem derived from PI 183967, and showed that it was a quantitative trait controlled by three major genes. QTL mapping identified five QTLs on Chr. 1, 3 and 6 using SSR markers. QTL gsb-s6.2 had the highest phenotypic variation of 22.7%, and was regarded as the major QTL with a physical distance of 1.9 Mb (Zhang et al. 2017). QTL mapping is an effective way to elucidate quantitative traits in plants. However, genes associated with resistance in PI 183967 have not been identified.
In this study, we fine-mapped and delimited our previously identified gsb-s6.2 locus to a 34 kb genomic region containing six candidate genes (Csa6G045200, Csa6G046200, Csa6G046210, Csa6G046220, Csa6G046230 and Csa6G046240). These genes have different functional annotations, encoding vesicle-associated membrane proteins, an AMMECR1 protein family, tumor-associated proteins, phosphate synthases, ATPase inhibitors, and MYB transcription factors, respectively (Table 2). Based on expression analysis and sequence alignment, we identified Csa6G046210 and Csa6G046240 were the main candidate genes underlying gsb-s6.2.
Csa6G046210 encodes an AMMECR1 family protein. We identified two nonsynonymous SNPs and one 21 bp InDel, which resulted in two amino acids alteration (Thr92Arg and Val200Glu) and the Indel of seven amino acids (Fig. 3). AMMECR1 has a conserved MMtag domain and may contain nuclear localization signals, so it might act as a signal molecule in the nucleus. AMMECR1 is one of the important genes of the contiguous gene deletion syndrome AMME in mammals (Thomas et al., 2010). Homologs of AMMECR1 in Arabidopsis, AT3G52220, encodes kinase phosphorylation protein and contains phosphorylation sites for several protein kinases. But its function in plants is not clear. Protein-protein interaction showed that the MMtag domain could interact with the WD40 repeat domain. WD-repeat proteins are involved in a variety of regulatory processes ranging from signal transduction and transcription regulation to cell cycle control and apoptosis. In wheat (Triticum aestivum L.), numerous TaWD40s were involved in response to biotic stresses and mainly expressed at the early stage of pathogen infection (Hu et al., 2018). Csa6G046210 showed a distinct expression pattern between two parents (P < 0.05). Therefore, we inspected its regulatory regions and found two significant InDels which caused deletions in two TATA-box elements (Fig. S1). As the core promoter element, the TATA box recruits the basal transcription machinery for transcription initiation. Further, a recent study found that the TATA box could influence promoter strength (Jores et al., 2021). We speculate that Csa6G046210 may play a role in GSB resistance and has a response to pathogen by regulating gene expression, however, we cannot exclude the changes of protein. More functional experiments need to be done, and to verify the functional mechanism of Csa6G046210.
Csa6G046240 encodes a transcription factor DIVARICATA, and showed the most significantly increased expression after inoculation among the candidate genes in the QTL interval. qRT-PCR analysis showed that Csa6G046240 was the most significantly up-regulated in both parental lines, but its expression levels were noticeably higher in GSB-susceptible parent ‘931’ compared to the GSB-resistant parent ‘PI 183967’ (Fig. 3e). We were unable to identify nonsynonymous variants in the Csa6G046240 coding sequences between two parents. Therefore, we speculated that Csa6G046240 maybe contain mutations in the promoter cis-regulatory elements. Many mutations in the promoter regions were discovered (Fig. 4), including three variants in AP2/ERF motifs, and another three variants were in MYB motifs. The AP2/ERF and MYB motifs might be part of the GSB resistance response to pathogen infection, by activating gene expression. The homologous gene in Arabidopsis encodes an R-R type MYB protein, which has a negative regulatory role in plant salt stress response and is essential for ABA signal transduction according to the description from GenBank records (AT5G04760). MYB transcription factors are widely distributed in plants, and interact with various transcription factors and play an important role in biotic and abiotic stresses (Ambawat et al., 2013). After pathogen infection, AtMYB30 triggers programmed cell death and a hypersensitive response by regulating hormone levels and specific gene expression (Daniel et al., 1999; Raffaele et al., 2006; Raffaele et al., 2008; Li et al., 2009). In our study, Heat shock proteins 70 (HSP70) are predicted to interact with CsDIV. Heat shock proteins are known to protect plants against abiotic and biotic stresses by maintaining protein homeostasis (Tutar et al., 2010; Zhang et al., 2015; Vierling, 1991; Sun et al., 2002; Wang et al., 2004). Thus, as a transcription factor, Csa6G046240 might modulate pathogen infection response by regulating downstream signaling pathways.
To determine the regulatory mechanisms that contribute to the GSB-resistance observed in this study, the two candidate genes Csa6G046210 and Csa6G046240 are being cloned and functionally analyzed. These candidate genes will lay the foundation for revealing the mechanism to GSB resistance and may be useful for marker-assisted selection in disease breeding in cucumber.