Legume-infecting Begomoviruses: Diversity and Host Interaction

Abstract

The legume yellow mosaic viruses (LYMVs) are members of the proposed sub-genus “Legumovirus” within the genus Begomovirus of the family Geminiviridae; single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. The legumoviruses are evolutionarily distinct from all other begomoviruses and are of interest for this reason as well as for the losses they cause to leguminous crops across southern Asia. There are four LYMVs (Mungbean yellow mosaic virus [MYMV], Mungbean yellow mosaic India virus [MYMIV], Dolichos yellow mosaic virus [DoYMV] and Horsegram yellow mosaic virus [HgYMV]) that have been shown to be responsible for yellow mosaic disease (YMD) of legumes across southern Asia. An analysis of the genetic diversity of LYMVs across Pakistan was conducted. Samples were collected from 11 districts across Pakistan and 48 full-length begomovirus components (25 DNA-A, 21 DNA-B) were cloned and sequenced in their entirety. Analysis of these sequences showed that MYMIV is the most prevalent causal agent of YMD in legume crops in Pakistan and shows phylogeographic segregation; no other virus species was shown to cause YMD of leguminous crops. MYMV, which is the major pathogen responsible for YMD of legumes in southern and western India, was also identified in Pakistan but this was identified only in a leguminous weed, Rhynchosia capitata. In addition a novel begomovirus, with less than 70% nucleotide sequence identity to all other begomoviruses, was isolated from another leguminous weed, Rhynchosia minima. This newly identified begomovirus was shown to belong to the LYMV cluster and was tentatively named Rhynchosia yellow mosaic virus (RhYMV). As well as the LYMV components, two virus species not commonly identified in legumes (Pedilanthus leaf curl virus [PedLCV] and Papaya leaf curl virus [PaLCuV]) as well as a betasatellite (Tobacco leaf curl betasatellite [TbLCB]) were isolated from some legumes infected with MYMIV and showing typical YMD symptoms. Constructs for the Agrobacterium-mediated inoculation of representative isolates of all begomovirus species and two isolates of MYMIV were produced. The MYMV was shown to infect blackgram, inducing very mild symptoms. RhYMV was shown to be infectious to some lines of soybean but not any of the other leguminous crops tested. The limited host range of these two viruses possibly explains their absence in crops. In contrast, two isolates of MYMIV, isolated from soybean (MYMIV-Sb) and mungbean (MYMIV-Mg) showed differing infectivities to legumes. The soybean isolate showed high levels of infectivity to soybean but low levels in blackgram, whereas the mungbean isolate was highly infectious to blackgram but poorly infectious to mungbean. This suggests that isolates of this virus are adapted to distinct hosts. None of the LYMVs examined was infectious to the non-legume Nicotiana benthamiana, a species which is commonly used as an experimentally host for all other dicot-infecting begomoviruses for which infectivity has been investigated. This is the first time this lack of infectivity to N. benthamiana has been reported. Similarly the viruses were not infectious to N. tabacum. The identification of a betasatellite in legumes is of grave concern due to the possibility of it increasing disease severity. TbLCB was shown to have the capacity to be maintained by MYMIV, MYMV and RhYMV, the first time a betasatellite has been shown to be trans-replicated by a LYMV, and to extend the host range of these viruses to N. benthamiana. Although DNA-B of all three viruses had some role to play in such infections (co-inoculation of DNA-B or expression of the DNA-B encoded MP under the control of the 35S promoter increased infectivity of MYMIV DNA-A and TbLCB from 60% to 100%), in the absence of DNA-B, TbLCB complemented the usual functions of DNA-Bs of all three viruses. This ability of TbLCB to complement DNA-B functions was shown to be a function of the only gene product encoded by betasatellites, βC1. Expression of TbLCB βC1 from PVX or transiently under the control of the 35S promoter allowed MYMIV to move systemically in N. benthamiana. However, when βC1 was expressed transiently using the 35S promoter, virus levels in systemically infected tissues were low and no symptoms ensued, suggesting that the βC1 function that assists MYMIV infection acts only at the site of inoculation and does not spread. The results obtained indicate that the lack of the infectivity of MYMIV to N. benthamiana is due to a lack of adaptation of the DNA-B-encoded products to this host. Thus when complemented by TbLCB, or by one of several monopartite begomoviruses (including PedLCV), MYMIV was able to efficiently spread systemically. In addition, plant host-defense mediated by RDR6 was shown to play a small role in limiting infection in N. benthamiana. However, silencing of this gene by VIGS did not allow MYMIV to induce a symptomatic infection. At this time the transformation of many legumes, particularly the grain legumes, is problematic, precluding the use of legume-transformation for the study of pathogen derived resistance to the LYMVs. Using a novel system, based upon the complementation of MYMIV movement using TbLCB, N. benthamiana was shown to potentially be a useful model host for such studies. Using transient expression of an antisense Rep gene construct, the infectivity of MYMIV (in the presence of TbLCB) was reduced by 90%. This indicates that RNAi may be a useful tool in reducing losses to LYMVs across Asia and that the betasatellite assisted infectivity system provides a means of selecting the most efficient constructs prior to efficient transformation protocols for local legume species becoming available.