Performance of New Muchamiel Tomato Lines with Virus Resistance Genes Grafted onto Two Commercial Rootstocks

Tomato landraces are regaining interest in Spain because their great fruit quality and value in popular gastronomy. Muchamiel is a traditional tomato variety grown in SE Spain that has been recently improved by the CIAGRO-UMH Tomato Breeding Group, resulting in several lines and hybrids with genetic resistances to virus and most of the original Muchamiel genome. In the current study, two hybrids and one pure line from CIAGRO-UMH and a commercial Muchamiel were grown under conventional conditions to evaluate three different grafting treatments: non-grafting and grafting onto the commercial Beaufort and Maxifort rootstocks. The yield parameters and fruit quality were assessed, and a sensory analysis was performed to evaluate the behavior of every scion/rootstock combination. Overall, significantly worse yield and fruit number in Maxifort-grafted plants were reported; as well as a slight reductions in SSC, fructose, and sucrose; and significant effects on few sensory traits. Instead, Beaufort-grafted plants showed no reduced yield, whereas no differences were reported between grafting treatments in fruit weight, TA, and acid profile, as well as in most of flavor and texture sensory parameters. These results suggest that Muchamiel/Beaufort combination could be suitable under unfavorable conditions, while Maxifort do not seem to provide agronomic nor quality benefits.

CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

Transgenic Elite Lines of Carioca Seeded Common Bean (Phaseolus Vulgaris L.) With Multiple Resistance to Viruses Reduce Cowpea Mild Mottle Virus Transmission

The most important viruses infecting common bean (Phaseolus vulgaris L.) in Brazil are BCMV, BGMV and CPMMV, the last two transmitted by the whitefly Bemisia tabaci, occurring simultaneously and causing severe yield losses. Genetically modified progenies of common bean, from carioca market class and multiple virus resistance (BCMV, BGMV and CPMMV), have been developed using conventional breeding and molecular tools. Agronomic performance and virus disease severity (VS) evaluated in two field trials, selected 39 elite progenies out of 477. Molecular analyses identified the presence of BCMV and BGMV resistance alleles in plants. CPMMV resistance was measured on mechanically inoculated plants using a VS scoring scale. Among the lowest VS average scores, five progenies showed resistance to BCMV, BGMV and CPMMV, and upright plant architecture, resistance to plant lodging and carioca market class grains, presenting potential to be developed into a new transgenic cultivar, with multiple virus resistance. Additionally, the resistant progenies may also contribute to reduce virus spread in the field, as they were a less efficient inoculum source of CPMMV in insect transmission assays.

Reprogramming of RNA silencing triggered by cucumber mosaic virus infection in Arabidopsis

Background RNA silencing has an important role mediating sequence-specific virus resistance in plants. The complex interaction of viruses with RNA silencing involves the loading of viral small interfering RNAs (vsiRNAs) into its host ARGONAUTE (AGO) proteins. As a side effect of their antiviral activity, vsiRNAs loading into AGO proteins can also mediate the silencing of endogenous genes. Here, we analyze at the genome-wide level both aspects of the interference of cucumber mosaic virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana. Results We observe CMV-derived vsiRNAs affect the levels of endogenous sRNA classes. Furthermore, we analyze the incorporation of vsiRNAs into AGO proteins with a described antiviral role and the viral suppressor of RNA silencing (VSR) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. Interestingly, vsiRNAs represent a substantial percentage of AGO-loaded sRNAs and displace other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affect the expression of the genes they derive from. Additionally, we analyze how vsiRNAs incorporate into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing, which allow us to identify vsiRNA-targeted genes genome-wide. Conclusions This work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.

Virus Yellows in sugar beet – possibilities to achieve virus resistance

Virus yellows in sugar beet is caused by different virus species. Monitoring has shown that Beet yellows virus (BYV), Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) are common and widespread, while Beet mosaic virus (BtMV) is less prevalent. The green peach aphid (Myzus persicae) is considered the main vector of these viruses. Sugar beet varieties with resistance or tolerance traits are currently not available to practical growers, therefore it is imperative to support breeding efforts with improved strategies to achieve virus resistance. For this purpose, a field test was established in which yield differences between susceptible and tolerant varieties can be generated by a 3% inoculation with BMYV-carrying aphids. A greenhouse bioassay has also been developed to distinguish susceptible and tolerant genotypes following BYV infection. Both assays pave the way for future use of natural resources such as wild forms and other breeding material to screen for virus resistance. In addition, molecular biology approaches are used to identify plant susceptibility factors of the plant-virus interaction, which will be knocked out via modern precision breeding methods to generate recessive virus resistance. Consequently, genotypes with naturally occurring mutations in the appropriate factors can be used for crossbreeding processes into elite breeding material.

Deciphering the Genetic Architecture of Plant Virus Resistance by GWAS, State of the Art and Potential Advances

Growing virus resistant varieties is a highly effective means to avoid yield loss due to infection by many types of virus. The challenge is to be able to detect resistance donors within plant species diversity and then quickly introduce alleles conferring resistance into elite genetic backgrounds. Until now, mainly monogenic forms of resistance with major effects have been introduced in crops. Polygenic resistance is harder to map and introduce in susceptible genetic backgrounds, but it is likely more durable. Genome wide association studies (GWAS) offer an opportunity to accelerate mapping of both monogenic and polygenic resistance, but have seldom been implemented and described in the plant–virus interaction context. Yet, all of the 48 plant–virus GWAS published so far have successfully mapped QTLs involved in plant virus resistance. In this review, we analyzed general and specific GWAS issues regarding plant virus resistance. We have identified and described several key steps throughout the GWAS pipeline, from diversity panel assembly to GWAS result analyses. Based on the 48 published articles, we analyzed the impact of each key step on the GWAS power and showcase several GWAS methods tailored to all types of viruses.

Development and Adoption of Genetically Engineered Plants for Virus Resistance: Advances, Opportunities and Challenges

Plant viruses cause yield losses to crops of agronomic and economic significance and are a challenge to the achievement of global food security. Although conventional plant breeding has played an important role in managing plant viral diseases, it will unlikely meet the challenges posed by the frequent emergence of novel and more virulent viral species or viral strains. Hence there is an urgent need to seek alternative strategies of virus control that can be more readily deployed to contain viral diseases. The discovery in the late 1980s that viral genes can be introduced into plants to engineer resistance to the cognate virus provided a new avenue for virus disease control. Subsequent advances in genomics and biotechnology have led to the refinement and expansion of genetic engineering (GE) strategies in crop improvement. Importantly, many of the drawbacks of conventional breeding, such as long lead times, inability or difficulty to cross fertilize, loss of desirable plant traits, are overcome by GE. Unfortunately, public skepticism towards genetically modified (GM) crops and other factors have dampened the early promise of GE efforts. These concerns are principally about the possible negative effects of transgenes to humans and animals, as well as to the environment. However, with regards to engineering for virus resistance, these risks are overstated given that most virus resistance engineering strategies involve transfer of viral genes or genomic segments to plants. These viral genomes are found in infected plant cells and have not been associated with any adverse effects in humans or animals. Thus, integrating antiviral genes of virus origin into plant genomes is hardly unnatural as suggested by GM crop skeptics. Moreover, advances in deep sequencing have resulted in the sequencing of large numbers of plant genomes and the revelation of widespread endogenization of viral genomes into plant genomes. This has raised the possibility that viral genome endogenization is part of an antiviral defense mechanism deployed by the plant during its evolutionary past. Thus, GM crops engineered for viral resistance would likely be acceptable to the public if regulatory policies were product-based (the North America regulatory model), as opposed to process-based. This review discusses some of the benefits to be gained from adopting GE for virus resistance, as well as the challenges that must be overcome to leverage this technology. Furthermore, regulatory policies impacting virus-resistant GM crops and some success cases of virus-resistant GM crops approved so far for cultivation are discussed.

Long-Term Efficacy and Safety of RNAi-Mediated Virus Resistance in ‘HoneySweet’ Plum

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. ‘HoneySweet’ was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. ‘HoneySweet’ has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in ‘HoneySweet,’ the nature and stability of its sRNA profile, and the potential health risks of consuming ‘HoneySweet’ plums. Graft-challenged ‘HoneySweet’ trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.

Membrane Trafficking Proteins: A New Target to Identify Resistance to Viruses in Plants

Replication cycles from most simple-stranded positive RNA viruses infecting plants involve endomembrane deformations. Recent published data revealed several interactions between viral proteins and plant proteins associated with vesicle formation and movement. These plant proteins belong to the COPI/II, SNARE, clathrin and ESCRT endomembrane trafficking mechanisms. In a few cases, variations of these plant proteins leading to virus resistance have been identified. In this review, we summarize all known interactions between these plant cell mechanisms and viruses and highlight strategies allowing fast identification of variant alleles for membrane-associated proteins.