CsMYB60 Confers Enhanced Resistance to Fusarium solani by Increasing Proanthocyanidin Biosynthesis in Cucumber.

Root rot caused by Fusarium solani is one of the most common fungal diseases in cucumber (Cucumis sativus). Proanthocyanidins (PAs) are known to play important roles in inhibiting the growth of phytopathogens. In addition, CsMYB60 is a known positive regulator of flavonol and PA biosynthesis in cucumber. However, it remains unclear that whether PAs can inhibit the growth of F. solani and whether CsMYB60 serves as a target gene for increasing resistance to phytopathogens in cucumber. In this study, we demonstrated that PAs (or their building block, catechin) could increase the resistance of cucumber seedlings to F. solani both in vitro and in vivo. The addition of catechins, or crude leaf extracts treated with different concentrations of catechins in culture medium, could significantly inhibit the hyphal growth of F. solani. On the other hand, cucumber seedlings treated with catechins showed higher resistance to F. solani than the seedlings of control group. Moreover, transgenic cucumber seedlings overexpressing CsMYB60, with the observed accumulation of PAs, were more resistant to F. solani than the nontransgenic siblings. Therefore, our results suggest that PAs (or catechin) can serve as a biological control agent to protect cucumber plants from the infection of F. solani. More importantly, CsMYB60 holds great promise as a target gene to confer disease resistance during the molecular breeding in cucumber.

CsMYB60 is a key regulator of flavonols and proanthocyanidans that determine the colour of fruit spines in cucumber.

Spine colour is an important fruit quality trait that influences the commercial value of cucumber (Cucumis sativus). However, little is known about the metabolites and the regulatory mechanisms of their biosynthesis in black spine varieties. In this study, we determined that the pigments of black spines are flavonoids, including flavonols and proanthocyanidins (PAs). We identified CsMYB60 as the best candidate for the previously identified B (Black spine) locus. Expression levels of CsMYB60 and the key genes involved in flavonoid biosynthesis were higher in black-spine inbred lines than that in white-spine lines at different developmental stages. The insertion of a Mutator-like element (CsMULE) in the second intron of CsMYB60 decreased its expression in a white-spine line. Transient overexpression assays indicated that CsMYB60 is a key regulatory gene and Cs4CL is a key structural gene in the pigmentation of black spines. In addition, the DNA methylation level in the CsMYB60 promoter was much lower in the black-spine line compared with white-spine line. The CsMULE insert may decrease the expression level of CsMYB60, causing hindered synthesis of flavonols and PAs in cucumber fruit spines.

CsMYB60 directly and indirectly activates structural genes to promote the biosynthesis of flavonols and proanthocyanidins in cucumber.

Flavonols and proanthocyanidins (PAs) are the main pigments in the black spines of cucumber (Cucumis sativus) fruit, and CsMYB60 is a key regulator of the biosynthesis of flavonols and PAs. However, in cucumber, the tissue distribution pattern of flavonols and PAs and the mechanism of their biosynthesis regulated by CsMYB60 remain unclear. In this study, we clarified the tissue-specific distribution of flavonoids and the unique transcriptional regulation of flavonoid biosynthesis in cucumber. CsMYB60 activated CsFLS and CsLAR by binding to their promoters and directly or indirectly promoted the expression of CsbHLH42, CsMYC1, CsWD40, and CsTATA-box binding protein, resulting in the formation of complexes of these four proteins to increase the expression of Cs4CL and interact with CsTATA-box binding protein to regulate the expression of CsCHS, thereby regulating the biosynthesis of flavonols and PAs in cucumber. Our data provide new insights into the molecular mechanism of flavonoid biosynthesis, which will facilitate molecular breeding to improve fruit quality in cucumber.

CsWRKY50 mediates defense responses to Pseudoperonospora cubensis infection in Cucumis sativus.

The cucumber (Cucumis sativus L.), an economically important vegetable crop, is often infected by Pseudoperonospora cubensis (P. cubensis), which results in inhibited growth and reduced yield. WRKY transcription factors (TFs) play critical roles in plant disease resistance. However, little is known about the function of WRKY TFs in cucumber downy mildew resistance. In this study, we reported that CsWRKY50, a cucumber WRKY subgroup Ⅱc TF localized in the nucleus, plays an important role in cucumber defense responses to downy mildew. In addition, several putative cis-acting elements involved in abiotic stress responsiveness were also identified in the CsWRKY50 promoter. Expression analysis revealed that CsWRKY50 can be induced by P. cubensis infection, abiotic stress and diverse signaling molecules. The overexpression of CsWRKY50 in cucumber enhanced the resistance of the plant to the fungal pathogen P. cubensis. In addition, less ROS accumulated in 35S:CsWRKY50 transgenic plants infected by the pathogen due to the higher expression levels of antioxidant enzymes. Importantly, after P. cubensis infection, the transcript levels of several hormone-related defense genes were also upregulated in transgenic plants, including SA- and JA-responsive genes and SA-synthesis genes. Collectively, our results indicate that CsWRKY50 positively regulates cucumber disease resistance to P. cubensis via multiple signaling pathways.