Climate change has favored the emergence of the Tomato leaf curl New Delhi virus (ToLCNDV) as a threat to melon production in the Mediterranean region. Deciphering the mechanisms regulating melon-ToLCNDV interactions is crucial in developing resistant varieties in the current climatic scenario. In this regard, DNA primase has recently been proposed as a recessive resistance gene for ToLCNDV. Here, we explored the molecular basis of ToLCNDV resistance in melon, focusing on the DNA-primase gene and the stress-responsive miR395. Using virus-induced gene silencing (VIGS) and transient expression assays, we show that DNA-primase silencing reduces ToLCNDV accumulation in susceptible plants, whereas overexpression increases the viral load in a resistant cultivar. Computational predictions, validated by transient expression analysis identified miR395 as a regulator of DNA-primase expression. Next, we found that adverse environmental conditions, such as salinity and drought, increase miR395 accumulation, downregulating DNA-primase and enhancing ToLCNDV resistance in susceptible melon cultivars. This study provides the first evidence that environmental conditions directly affect geminivirus infection dynamics via miR395-mediated DNA-primase regulation. These findings underscore the potential of targeting DNA-primase for breeding ToLCNDV-resistant melon varieties and highlight the environment influence on virus-host interactions, offering insights for sustainable disease management in crops.

Climate change has been associated with a higher incidence of combined adverse environmental conditions that can promote a significant decrease in crop productivity. However, knowledge on how a combination of stresses might affect plant development is still scarce. MicroRNAs (miRNAs) have been proposed as potential targets for improving crop-productivity. Here, we have combined deep-sequencing, computational characterization of responsive miRNAs and validation of their regulatory role in a comprehensive analysis of melon’s response to several combinations of four stresses (cold, salinity, short day, and infection with a fungus). Twenty-two miRNA families responding to double and/or triple stresses were identified. The regulatory role of the differentially expressed miRNAs was validated by quantitative measurements of the expression of the corresponding target genes. A high proportion (ca. 60%) of these families (mainly highly conserved miRNAs targeting transcription factors) showed a non-additive response to multiple stresses in comparison with that observed under each one of the stresses individually. Among those miRNAs showing non-additive response to stress-combinations, most interactions were negative suggesting the existence of functional convergence in the miRNA-mediated response to combined stresses. Taken together, our results provide compelling evidences that the response to combined stresses cannot be easily predicted from the study individual stresses.

Carotenoids and tocopherols are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. Here, we demonstrate an alternative biotechnological strategy for carotenoid and tocopherol fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated in zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and β-carotene (pro-vitamin A), lutein and phytoene, in both rind and flesh. Considerably higher accumulation of α- and γ-tocopherol was also detected, particularly in fruit rind. Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.