Gene structure and protein motif analysis of the CsCSE family
Differences in gene structure and the conservation of protein motifs may have important evolutionary implications (Xia et al. 2022). To clarify the structure of CsCSE genes, we analyzed the UTRs, CDSs, and introns of CsCSE genes based on the phylogenetic relationships among members of this family (Fig. 2a and c). The structure of CsCSE genes was variable; the number of UTRs ranged from two to four, the number of CDSs ranged from one to eight, and the number of introns ranged from zero to nine (Fig. 2c). CsCSE1, CsCSE4, and CsCSE6 contained the same number of UTRs (two), CDSs (eight), and introns (seven), yet they belonged to different clades. The same pattern was also observed in CsCSE2 and CsCSE3, both of which contained two UTRs, one CDS, and no introns (Fig. 2a, c). Moreover, the numbers of UTRs, CDSs, and introns of these two genes were the lowest in this family, and the greatest numbers of UTRs, CDSs, and introns were observed in CsCSE8, which contained four UTRs, eight CDSs, and nine introns. In addition to differences in the number of different gene components, the lengths of each gene component varied among CsCSE family members. The longest UTR was 1,033 bp (CsCSE7), and the shortest UTR was only 53 bp (CsCSE8). The lengths of CDSs ranged from 3 bp (CsCSE1) to 1,002 bp (CsCSE7). The longest and shortest introns were observed in the CsCSE4 gene (3,862 bp and 74 bp, respectively) (Fig. 2c). Differences in the length and number of gene components might underlie the formation of various functional motifs.
To further explore the functional characteristics of CsCSE proteins, the structural motifs of CsCSE proteins were evaluated using the online website MEME, and the results were visualized using TBtools software (Fig. 2b and Supplementary Table 2). A total of 10 conserved motifs were identified in the CsCSE family and named motif 1 to motif 10. No member of the CsCSE family had all 10 conserved motifs, and the number of motifs ranged from six (CsCSE3) to nine (CsCSE2 and CsCSE8). The structure of CsCSE2 and CsCSE3 was similar, but they differed in the number of motifs, which might stem from the fact that these two proteins were placed in different clades (Fig. 2). Motifs 1, 2, 3, and 4 were highly conserved in the CsCSE family; these were considered the characteristic motifs of CsCSE given that they were present in all members of this family. In addition, some motifs were only present in certain proteins, such as motif 9, which was only identified in CsCSE6 and CsCSE8, and motif 10 was only identified in CsCSE2 and CsCSE3 (Fig. 2b). CsCSE1, CsCSE4, and CsCSE5 contained the same motifs (motifs 1, 2, 3, 4, 5, 6, 7, and 8), suggesting that these three proteins may perform the same function. They were located in the same or similar clades, which reflects the positive relationship between heredity and function, and suggests that the topology of the phylogenetic tree is accurate (Fig. 2a, b). Furthermore, CsCSE2 and CsCSE8 contained the greatest number of conserved motifs and may have additional biological functions; however, further study is needed to confirm this possibility (Fig. 2b).
Cis -acting element analysis of CsCSE promoters
Cis-acting elements in gene promoter regions mediate responses to plant growth and stress by regulating the expression of genes (Walther et al. 2007). To clarify the expression and regulatory characteristics of CsCSE genes and identify their potential biological functions, we used the PlantCARE database to analyze the cis-acting elements in the upstream 2,000-bp promoter sequences of CsCSE genes. Various types of typical cis-acting elements were identified, including hormone-responsive elements (salicylic acid-responsive, MeJA-responsive, abscisic acid-responsive, gibberellin-responsive, and auxin-responsive elements), stress-responsive elements (defense and stress-responsive as well as low temperature-responsive elements), growth and development-related elements (seed-specific regulation, anaerobic induction, and zein metabolism regulation), and transcription factor-related elements (MYB and MYBHv1 binding site) (Fig. 3 and Supplementary Table 3). Anaerobic induction and MYB elements were most widely distributed in the CsCSE gene family (75% of CsCSE genes), with the exception of CsCSE6 (lack of anaerobic induction), CsCSE8 (lack of MYB), and CsCSE1 (lack of both). A total of 62.5% of CsCSE genes contained salicylic acid-responsive, MeJA-responsive, and zein metabolism regulatory elements, and 50% of CsCSE genes contained abscisic acid-responsive, gibberellin-responsive, and defense and stress-responsive elements. In addition, low temperature-responsive and MYBHv1-binding site elements were detected in CsCSE4 and CsCSE8, and an auxin-responsive element was also detected in CsCSE8. A seed-specific regulatory element was identified in CsCSE1 (Fig. 3). Overall, the presence of these functional elements suggests that the expression of CsCSE genes might be induced by a variety of hormones and that they are involved in specific growth, development, and signaling pathways as well as stress responses, thereby mediating the resistance of cucumber plants to various environmental stresses during their growth.
Chromosome distribution and collinearity analysis of CsCSE genes
The positions of CsCSE genes in the cucumber genome were visualized using TBtools software. As shown in Fig. 4, eight CsCSE genes were unevenly distributed on chromosomes 1, 3, 4, and 5. Chromosome 5 had the greatest number of CsCSE genes: CsCSE5, CsCSE6, CsCSE7, and CsCSE8. Two CsCSE genes (CsCSE1 and CsCSE2) were present on chromosome 1, only one CsCSE gene was present on chromosomes 3 and 4 (CsCSE3 and CsCSE4, respectively), and no CsCSE genes were present on chromosomes 2, 6, and 7. To explore the evolutionary history of CsCSE genes, we detected duplication events in members of this gene family. However, no tandem duplication and fragment duplication events were detected in CsCSE genes, and this reflects the evolutionary conservation of CsCSE genes within species (Fig. 4).
Collinear relationships among species provide insights into the evolution of gene families and gene functions (Yao et al. 2022). Collinearity analysis was performed on the genomes of cucumber and five other plants, A. thaliana, barrel medic (M. truncatula), poplar (P. trichocarpa), corn (Z. mays), and wax gourd (B. hispida), to further elucidate the evolutionary history of CsCSE genes (Fig. 5 and Supplementary Table 4). Collinear pairs of CSE genes between cucumber and P. trichocarpa were the most common (seven), followed by A. thaliana (six), B. hispida (six), M. truncatula (five), and Z. mays (three). A total of six CsCSE genes displayed collinear relationships with CSE genes in B. hispida, followed by M. truncatula (four), P. trichocarpa (four), A. thaliana (three), and Z. mays (three). The number of collinear pairs of CSE genes identified among various species was not completely consistent with the corresponding number of CsCSE genes because the same CsCSE gene might match with different CSE genes in the same species. For example, between cucumber and A. thaliana, XM_004146542.3 (CsCSE2) showed collinearity with both NM_104154.4 and NM_100982.3, XM_004152315.3 (CsCSE4) showed collinearity with both NM_130331.4 and NM_116151.4, and XM_004147661.3 (CsCSE5) showed collinearity with both NM_129497.4 and NM_115376.2. The same pattern was observed in comparisons of the genomes of cucumber and M. truncatula, as well as cucumber and P. trichocarpa. Moreover, collinear gene pairs of the three cucumber CsCSE members, CsCSE2, CsCSE4, and CsCSE5, were detected in all five species. The above results reveal the evolutionary differentiation of CSE genes between species. Collinear gene pairs of XM_004153438.3 (CsCSE1) and XM_004147950.3 (CsCSE3) were only detected between cucumber and B. hispida. The formation of these species-specific collinear gene pairs might be associated with the evolutionary mechanism of Cucurbitaceae plants, which indicates that these two genes were highly conserved during the evolution of cucumber and B. hispida. Additionally, CsCSE genes, with the exception of CsCSE6 and CsCSE8, could be matched with their corresponding CSE genes in B. hispida, which reflects the high homology of CSE genes during the evolution of Cucurbitaceae.
Expression patterns of CsCSE genes in cucumber varieties resistant and susceptible to P. xanthii and C. cassiicola infection
The expression patterns of genes under stress conditions usually reflect their potential functions in stress responses (Zhu et al. 2021). To investigate whether members of the CsCSE family are involved in the defense response of cucumber to pathogens, we evaluated the expression characteristics of eight CsCSE genes in B21-a-2-1-2 (resistant) and B21-a-2-2-2 (susceptible) leaves inoculated with P. xanthii as well as JinYou 35 (resistant) and XinTaiMiCi (susceptible) leaves vaccinated with C. cassiicola at 0, 6, 12, 24, 72, and 144 hpi (Fig. 6). The expression patterns of these CsCSE genes under infection of the two pathogens were diverse, but some similarities were observed. All eight genes were significantly differentially expressed in resistant and susceptible cucumbers infected with P. xanthii and C. cassiicola at various time points, and changes in expression were observed, which reflects their diverse functional roles in responses to pathogen stress.
Under P. xanthii infection, the expression of CsCSE5 was higher in resistant strains than in susceptible strains. However, the opposite patterns were observed for CsCSE2 and CsCSE6; that is, their expression levels were significantly lower in resistant strains than in susceptible strains. Therefore, we speculated that these two groups of CsCSE genes may be involved in the response of cucumber to attack by P. xanthii but have opposite regulatory roles. The expression peak of CsCSE5 was observed at 144 hpi in both resistant and susceptible strains, and its expression level was relatively low and stable in the early stage of P. xanthii infection, suggesting that CsCSE5 may be involved in regulating cucumber defenses during the late stage of P. xanthii stress. In contrast, the expression peak of CsCSE6 was observed at 0 hpi, and its expression was markedly down-regulated and remained low under P. xanthii infection. The expression of CsCSE1, CsCSE3, and CsCSE8 in susceptible strains also peaked at 144 hpi; unlike CsCSE5 however, the expression levels of these three CsCSE genes remained low in resistant strains. The expression of CsCSE4 fluctuated greatly during P. xanthii infection, and the opposite expression patterns were observed in resistant and susceptible strains. Specifically, the expression of CsCSE4 first decreased and then increased in resistant strains, but first increased and then decreased in susceptible strains, which means that this gene might play an important role in all stages of P. xanthii transmission (Fig. 6a).
In addition, the expression patterns of eight CsCSE genes under C. cassiicola infection were also diverse. The expression of CsCSE1, CsCSE4, and CsCSE8 was significantly lower in resistant strains than in susceptible strains, indicating that these three genes might play a negative regulatory role in the resistance of cucumber to C. cassiicola. In contrast, the expression pattern of CsCSE6 was opposite to that of these genes; we speculate that CsCSE6 might positively regulate the resistance of cucumber to C. cassiicola. The expression of all eight genes peaked at 72 hpi in resistant or susceptible strains, and the most pronounced differences in the expression of CsCSE2, CsCSE6, and CsCSE7 in resistant and susceptible strains were observed at this time point, suggesting that this timepoint might be critically important for the resistance of CsCSE genes to C. cassiicola. Aside from CsCSE1, only CsCSE5 was differentially expressed at 144 hpi, indicating that this gene may play a role in regulating the tolerance of cucumber to C. cassiicola during the later stages of infection (Fig. 6b).
In conclusion, all eight CsCSE genes may mediate cucumber–P. xanthii and cucumber–C. cassiicola interactions. The function of the CsCSE5 gene was studied in light of the fact that its differential expression was most pronounced in resistant and susceptible cucumber leaves infected with P. xanthii and C. cassiicola and the fact that it shows highest homology with CsCSE1, which has been shown to play a positive regulatory role in the resistance of cucumber to P. xanthii (Yu et al. 2022) (Fig. 6 and Fig. 2a).
Silencing of CsCSE5 attenuated the resistance of cucumber to P. xanthii and C. cassiicola
To characterize the function of CsCSE5 in mediating the tolerance of cucumber to P. xanthii and C. cassiicola, we generated plants with the CsCSE5 gene knocked down using a CGMMV-based VIGS approach and investigated the effect of CsCSE5 silencing on cucumber disease resistance. As previously mentioned, the pV190 vector modified by CGMMV can be used to induce gene silencing in cucurbit plants (Liu et al. 2020). In this study, a 289-bp specific fragment of the CsCSE5 gene was inserted into the pV190 vector, which yielded the pV190-CsCSE5 recombinant vector (Fig. 7a). The expression of CsCSE5 was significantly reduced in pV190-CsCSE5 plants relative to pV190 plants (Fig. 7b). These results indicated that CsCSE5-silenced cucumber plants had been successfully acquired.
The leaves of these CsCSE5-silenced and control cucumbers were vaccinated with P. xanthii and C. cassiicola to evaluate powdery mildew (PM) and target leaf spot (TLS) disease incidence, respectively. PM and TLS symptoms in both pV190-CsCSE5 and pV190 plants were observed at 7 dpi, but the PM and TLS symptoms of pV190-CsCSE5 cucumbers were particularly severe (Fig. 7c and e). Pictures of P. xanthii hyphae stained with CBB obtained using a microscope indicated that hyphal density was markedly greater in pV190-CsCSE5 cucumbers than in pV190 cucumbers, suggesting that CsCSE5-silencing facilitated the growth of P. xanthii hyphae (Fig. 7d). Moreover, at least 60 pV190-CsCSE5 and pV190 plants infected with P. xanthii and C. cassiicola were used to calculate the DI. The DI of pV190-CsCSE5 plants was higher than that of pV190 plants in both P. xanthii and C. cassiicola (Fig. 7c and e). In sum, the silencing of CsCSE5 in cucumber attenuated resistance to P. xanthii and C. cassiicola, and CsCSE5 was required for the defense of cucumbers against pathogens.