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.

Genome editing and gene expression engineering using CRISPR-Cas systems in plants usually rely on labor-intensive tissue culture approaches to generate stably transformed plants that express the components of the reaction. Viral vectors have demonstrated to be a quick and effective alternative to express multiple guide RNAs, DNA templates for homologous recombination, and even Cas nucleases. Here we have developed an improved vector system based on tobacco rattle virus (TRV) to simplify logistics in genome editing and gene silencing approaches. The new system consists in a single Agrobacterium tumefaciens clone co-transformed with two compatible mini binary vectors from which TRV RNA1 and an engineered version of TRV RNA2 are expressed. Sequences of recombinant proteins, gene fragments for virus-induced gene silencing (VIGS) or guide RNAs can be easily inserted by one-step digestion-ligation and homology-based cloning methods in the RNA2 plasmid to produce vectors with a size substantially smaller than usual. Using this new one-Agrobacterium TRV mini vector system, we show robust VIGS of an endogenous host gene after infiltration of bacterial suspensions at low optical densities, and efficient production of recombinant proteins in Nicotiana benthamiana. Most importantly, we also show highly efficient heritable genome editing in more than half of the seedling originating from inoculated N. benthamiana plants that express Cas9.

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.