Molecular Pathogens, 2025, Vol.16, No.4, 193-206 http://microbescipublisher.com/index.php/mp 202 7.3 Regulatory network of disease-resistant gene CsMLO and related transcription factors In the genetic regulation of cucumber resistance to powdery mildew, susceptible gene CsMLOand its regulatory network are a research hotspot. Members of the cucumber MLO gene family (such as CsMLO1, CsMLO8, CsMLO11, etc.) promote pathogen invasion during powdery mildew infection and are the key factors that lead to sensory diseases. Functional studies of CsMLO1 have found that knockdown of this gene (e.g., through CRISPR/Cas9-mediated mutations) can confer durable resistance to powdery mildew, but may be accompanied by some growth and developmental effects such as tendril abnormalities (Figure 3) (Shnaider et al., 2022; Ma et al., 2024). Further research will focus on the regulatory network behind the MLO-mediated sensory pathogenesis. In addition to MLO itself, there are some important transcription factors in the anti-powder mildew regulatory network. WRKY and NAC are involved in early responses to powdery mildew infection. For example, the aforementioned CsWRKY50 plays a role in downy mildew resistance and is also considered to contribute to powdery mildew resistance: experiments have shown that cucumbers overexpressing CsWRKY50 not only have enhanced downy mildew resistance, but also have reduced the incidence of powdery mildew. It is speculated that WRKY50 may produce broad-spectrum disease-resistant effects by increasing gene expression of SA pathway. CsWRKY6, which belongs to the WRKY family, is highly expressed in cucumber epidermal cells and is speculated to be involved in the defense of the invasion stage of powdery white bacteria. In terms of NAC transcription factors, studies have noticed that upregulation of a NAC gene called SHN1 in cucumber (wax synthesis-related) is positively correlated with powdery mildew resistance, and may alleviate infection by increasing the thickness of the epidermal wax. In addition, ERF1 transcription factors downstream of the JA/ET pathway can enhance necrotic pathogen resistance as described previously, but are not a major regulatory factor for powdery mildew. However, ORA59 in the ERF class has been found to interact with WRKY target genes, which may indirectly affect the SA pathway. 8 Applications and Outlook 8.1 Application prospects of molecular breeding in cucumber disease-resistance improvement The rapid development of molecular breeding technology has injected new impetus into the cultivation of cucumber disease-resistant varieties. Traditional cucumber disease-resistant breeding mainly relies on the introduction of disease-resistant genes from wild species or other varieties, and obtain disease-resistant strains through hybrid selection. However, traditional methods are long cycles and are limited by available anti-sources. Modern molecular breeding includes gene marker assisted selection (MAS), genome-wide selection (GS), and molecular design breeding, which can greatly improve the efficiency of disease-resistant breeding. For crops like cucumbers with rich self-incompatible lines, marker-assisted selection has been initially used in disease-resistant breeding. For diseases such as black star disease, downy mildew, cucumber mosaic virus, molecular markers linked to main-effect resistance genes or QTLs have been developed, which can be used to early screen for offspring carrying resistance alleles. This allows breeders to select target plants through DNA testing without inoculating pathogens, greatly improving selection accuracy and speed. As more and more disease-resistant genes are cloned, a "combination package" of molecular markers for cucumber disease-resistant genes can be established in the future, and multiple disease resistances can be selected at the same time, thereby cultivating varieties with multiple resistances. 8.2 The potential of CRISPR/Cas technology in the analysis and utilization of disease-resistant gene functions The rise of CRISPR/Cas gene editing technology has brought revolutionary tools to genetic improvement of plants with disease resistance. Compared with traditional breeding and transgenic technologies, CRISPR/Cas can accurately modify the crop genome, which can be used to functionally verify the disease-resistant gene mechanism, and can be used to directly improve crop resistance status. In the field of cucumber disease resistance, CRISPR/Cas has begun to show its power. A typical application is to target knockout of susceptible genes to obtain disease resistance.
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