Engineering resistance against geminiviruses: A review of suppressed natural defenses and the use of RNAi and the CRISPR/Cas system

Highlights

• Manipulating NIK1-mediated antiviral signaling for resistance to begomovirus.

• RNA silencing-based defenses engineered against geminivirus.

• Resistance against geminiviruses via the CRISPR/Cas9 system.

Abstract

The Geminiviridae family is one of the most successful and largest families of plant viruses that infect a large variety of important dicotyledonous and monocotyledonous crops and cause significant yield losses worldwide. This broad spectrum of host range is only possible because geminiviruses have evolved sophisticated strategies to overcome the arsenal of antiviral defenses in such diverse plant species. In addition, geminiviruses evolve rapidly through recombination and pseudo-recombination to naturally create a great diversity of virus species with divergent genome sequences giving the virus an advantage over the host recognition system. Therefore, it is not surprising that efficient molecular strategies to combat geminivirus infection under open field conditions have not been fully addressed. In this review, we present the anti-geminiviral arsenal of plant defenses, the evolved virulence strategies of geminiviruses to overcome these plant defenses and the most recent strategies that have been engineered for transgenic resistance. Although, the in vitro reactivation of suppressed natural defenses as well as the use of RNAi and CRISPR/Cas systems hold the potential for achieving broad-range resistance and/or immunity, potential drawbacks have been associated with each case.

Introduction

Geminiviridae is a large family of plant viruses characterized by the unique morphology of their icosahedral geminated capsids and divided into nine genera, Becurtovirus, Begomovirus, Capulavirus, Curtovirus, Grablovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus, which are classified based on genome structure (monopartite or bipartite), insect vector (whitefly, treehopper, leafhopper or aphid), host (monocotyledonous or dicotyledonous) and phylogenetic origin of the species [1]. The Geminiviridae family encompasses broadly distributed pathogenic species that infect a large variety of essential crops and pose a severe constraint to crop and vegetable productivity and hence food security. These include maize, bean, cotton, tomato, cassava, pepper, okra, potato, cabbage, squash, and others. The symptoms of diseases caused by geminiviruses typically consist of leaf curling, mosaic, mottle, vein yellowing, or leaf yellowing as well as chlorosis, crumpling, and rugosity. Infected plants are often stunted, particularly when infected early in development, and yields can be substantially reduced. The incidence and severity of diseases caused by geminiviruses have increased considerably for the past 20 years [2,3], due to high levels of mutations, recombination and rearrangement of viral genomes, leading to a concomitant increase in the diversity of this family [[4], [5], [6]]. Current climate changes are expected to alter more the insect vector distribution throughout the globe, posing a significant threat to agriculture worldwide. Currently, two species of geminiviruses, belonging to the Begomovirus genus, the monopartite Tomato yellow leaf curl virus (TYLCV) and the bipartite African cassava mosaic virus (ACMV), are included in the top ten list for economically important plant viruses [7]. ACMV causes the most devastating disease in cassava, the cassava mosaic disease, and impact the subsistence of African population considerably. The incidence of tomato yellow leaf curl diseases has increased considerably around the globe and currently TYLCV represents a constraint to agriculture in North and Central Americas, Africa, Asia, Europe and Middle East. Bean golden mosaic virus (BGMV) represents a serious threat to the bean production in Brazil. In South America, the large diversity of tomato-infecting begomoviruses, which has emerged with increased pathogenicity, has made broad range resistance a challenging agronomic trait to be achieved. Thus, it is not surprising the existence in the literature of innumerous attempts for the development of pathogen-derived resistance by expressing begomovirus DNA sequences in transgenic plants. In fact, transgenic expression of defective AC1 protein, antisense AC1 (replication-associated protein) gene, defective movement protein, defective viral DNA and coat protein has been demonstrated to attenuate geminivirus infection [[8], [9], [10], [11], [12], [13]]. However, none of the previously described transgenic plants had an immune, resistance, or tolerance responses, but rather they displayed at most a delayed infection rate and attenuated symptoms in a narrow spectrum fashion.

On the other hand, transgenic tomato lines expressing peptide aptamers, which bind efficiently to and inhibit the begomovirus replication protein (Rep), have been shown to display enhanced tolerance to TYLCV or Tomato mottle virus (ToMoV) [14]. Likewise, expression of the single-stranded DNA binding protein virE2 from Agrobacterium in tobacco has been shown to reduce Mungbean yellow mosaic virus (MYMV) DNA accumulation, although the spectrum of the resistance has not been established [15]. Nevertheless, in none of these studies, the efficiency of the acquired resistance has been evaluated under open field conditions and pressure of high-density population of the insect vector. As exceptions, transgenic lines expressing a siRNA for Rep gene silencing from BGMV [16], and TYLCV [17] are immune to these begomoviruses under field conditions. However, the spectrum of these siRNA-based acquired resistance against BGMV- and TYLCV-related begomoviruses has not been determined.

Strategies for RNAi-derived resistance targeting different viral sequences have been extended to include resistance to Sri Lankan cassava mosaic virus (SLCMV) [18]; TYLCV [19]; Chilli leaf curl virus (ChiLCV) [20]; Mungbean yellow mosaic India virus (MYMIV) [21]; Croton yellow vein mosaic virus (CrYVMV) [22]; Cotton leaf curl virus (CLCuRV) [[23], [24], [25]]. Polycistronic artificial miRNA has also been used to generate resistance to insect-mediated Wheat dwarf virus (WDV) infection [26]. Genome editing has also been used to create plant’s resistance to geminiviruses by efficiently targeting specific regions in the viral genome using the clustered regularly interspaced palindromic repeats-associated nuclease (CRISPR-Cas) systems. These studies include the use of gene-editing technology to create resistant plants to Beet severe curly top virus (BSCTV) [27,28]; Bean yellow dwarf virus (BeYDV) [29], and TLYCV [[30], [31], [32]]. Although these results are promising and immunity has been achieved, potential pitfalls related to off-target effects and rapid evolution of viral variants that may escape recognition by the CRISPR/Cas9 system are still a matter of debate [33,34].

The genome of geminiviruses may be either mono or bipartite and is encapsidated as circular single-stranded DNA that is converted into double-stranded DNA in the nuclei of infected cells [35]. Geminiviruses depend extensively on the host replication machinery for viral DNA replication and interact with a large variety of host proteins during infection, reprogramming the cell cycle of infected cells to induce viral DNA and host chromosome replication [35]. Furthermore, the geminiviruses are capable of altering gene expression profiles, inhibiting cell death pathways, altering the macromolecule trafficking, interfering in cell signaling and protein turnover to redirect or block host defenses and hormone signaling, [35,36]. This review describes briefly some mechanisms of anti-geminiviral defenses of plants, the virulence strategies to counteract the host defenses, and primarily the most recent strategies, which have been used to engineer plant resistance against geminiviruses, their development and perspectives.