Construction of an infectious full-length cDNA clone of a melon yellowing-associated virus from Brazil

Melon yellowing-associated virus (MYaV), a member of the genus Carlavirus (family Betaflexiviridae), is a positive-sense, single-stranded RNA virus with a poly(A) tail. It is thought to be one of the causative agents of severe yellowing disease in melon "Amarelão do meloeiro" (in Portuguese) in northeastern Brazil and is transmitted by the whitefly Bemisia tabaci. In the field, MYaV frequently occurs in mixed infections with the recombinant form of cucurbit aphid-borne yellows virus (CABYV), contributing to the disease complex of melon severe yellowing disease. Considering its impact, the inability of virus transmission by mechanical inoculation, and the lack of molecular tools to study this pathogen, we constructed a full-length infectious cDNA clone of MYaV using Gibson Assembly technology. The genome was amplified in two overlapping fragments spanning the 5' and 3' regions, cloned in the binary vector pJL89 and subsequently introduced to Agrobacterium tumefaciens strain GV3101. Agroinoculation with this construct revealed that the MYaV clone was infectious in melon, cucumber (Cucumis sativus), West Indian gherkin (Cucumis anguria), and watermelon (Citrullus lanatus). Notably, infections in watermelon and West Indian gherkin were asymptomatic, a finding of epidemiological relevance since these crops may act as unnoticed inoculum sources in the field for melon crops. In contrast, no infection was detected in squash (Cucurbita maxima) or zucchini (Cucurbita pepo). This infectious clone provides not only a valuable tool to advance studies on MYaV and virus-host interactions but also an essential resource for breeding programs aimed at developing resistant melon varieties.