Further characterization and detection of pineapple mealybug wilt associated viruses (PMWAVs)

Abstract

Mealybug wilt of pineapple (MWP) is one of the most destructive diseases of pineapple worldwide. A complex of pineapple mealybug wilt associated viruses (PMWaVs), all of which belong to the genus Ampelovirus (Family: Closteroviridae) have been identified in pineapple growing regions throughout the world. Three of these viruses, PMWaV-1, PMWaV-2, PMWaV-3 have been characterized. The disease etiology of MWP is still not completely resolved. To better understand the complex etiology of mealybug wilt of pineapple (MWP), it is essential that the genomes of PMWaVs be functionally characterized. In Hawaii, PMWaV-2 has an important role in the etiology of MWP, causing severe wilt symptoms and yield reductions in the presence of mealybug feeding, whereas infection by PMWaV-1 alone, with or without mealybug feeding, may cause some yield reduction but does not produce wilt symptoms. Higher plants use RNA silencing to defend against viral infections. As a counter defense, plant viruses have evolved proteins that suppress RNA silencing. In this study selected open reading frames (ORFs) of PMWaV-1 and PMWaV-2 were screened for their local and systemic suppressor activities in Agrobacterium-mediated transient assays using green fluorescent protein (GFP) in Nicotiana benthamiana. Results indicate that PMWaV-2 utilizes a multiplecomponent RNA silencing suppression mechanism. Two of the encoded proteins analyzed, p20 and CP, target both local and systemic silencing in N. benthamiana, whereas p22 and CPd targets only systemic silencing. In the related virus PMWaV-1, we found that only one of the encoded proteins analyzed, p61, had systemic suppressor activity. Of all the proteins tested from both viruses, only PMWaV-2 p20 suppressed local silencing induced by doublestranded (ds) RNA, but only when low levels of inducing dsRNA were used. None of the proteins analyzed showed an ability to interfere with the short distance systemic spread of silencing. We examined the mechanism of systemic suppression activity by investigating the effect of PMWaV-2-encoded p20 and CP proteins on secondary siRNAs. Our results suggest that PMWaV-2 p20 and CP (and probably other proteins) block the systemic silencing signal by repressing production of secondary siRNAs. We also demonstrate that PMWaV-2 p20 and p22 enhanced Potato virus X pathogenicity in N. benthamiana. The construction of a full-length and less than full length PMWaV-1 clone has been reported. The toxicity of certain region of the cloned PMWaV-1 genome in E. coli induced lethal mutations and re-arrangements, which might be the reason for its non-infectivity in N. benthamiana. A cell-free method, Circular polymerase extension cloning (CPEC) of creating infectious clone has been adapted successfully for the first time for a Potato virus X, plant virus. However technical refinement would require the making of an infectious clone of PMWaV-1. Considering the importance of PMWaV-2 in the disease etiology of MWP, a highly sensitive detection assay using a single closed tube nested PCR technique has been developed which can be used to detect very low PMWaV-2 titer in diseased plants and insects. The assay is made more flexible by inclusion of a Taqman® probe to make the assay quantitative.