Summary
The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/tpj.13446

High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.

Transgenic Bt maize that produces less than a high-dose has been widely adopted and presents considerable insect resistance management (IRM) challenges. Western corn rootworm, Diabrotica virgifera virgifera LeConte, has rapidly evolved resistance to Bt maize in the field, leading to local loss of efficacy for some corn rootworm Bt maize events. Documenting and responding to this resistance has been complicated by a lack of rapid diagnostic bioassays and by regulatory triggers that hinder timely and effective management responses. These failures are of great concern to the scientific and agricultural community. Specific challenges posed by western corn rootworm resistance to Bt maize, and more general concerns around Bt crops that produce less than a high-dose of Bt toxin, have caused uncertainty around current IRM protocols. More than 15 years of experience with IRM has shown that high-dose and refuge-based IRM is not applicable to Bt crops that produce less than a high-dose. Adaptive IRM approaches and pro-active, integrated IRM-pest management strategies are needed and should be in place before release of new technologies that produce less than a high-dose. We suggest changes in IRM strategies to preserve the utility of corn rootworm Bt maize by 1) targeting local resistance management earlier in the sequence of responses to resistance and 2) developing area-wide criteria to address widespread economic losses. We also favor consideration of policies and programs to counteract economic forces that are contributing to rapid resistance evolution.

/pdf/early-detection-and-mitigation-of-resistance-to-bt-maize-by-12f16ttond.pdf

Early Detection and Mitigation of Resistance to Bt Maize by Western Corn Rootworm (Coleoptera: Chrysomelidae)

Knowledge of how climate change is likely to influence future virus disease epidemics in cultivated plants and natural vegetation is of great importance to both global food security and natural ecosystems. However, obtaining such knowledge is hampered by the complex effects of climate alterations on the behavior of diverse types of vectors and the ease by which previously unknown viruses can emerge. A review written in 2011 provided a comprehensive analysis of available data on the effects of climate change on virus disease epidemics worldwide. This review summarizes its findings and those of two earlier climate change reviews and focuses on describing research published on the subject since 2011. It describes the likely effects of the full range of direct and indirect climate change parameters on hosts, viruses and vectors, virus control prospects, and the many information gaps and deficiencies. Recently, there has been encouraging progress in understanding the likely effects of some climate change parameters, especially over the effects of elevated CO2, temperature, and rainfall-related parameters, upon a small number of important plant viruses and several key insect vectors, especially aphids. However, much more research needs to be done to prepare for an era of (i) increasingly severe virus epidemics and (ii) increasing difficulties in controlling them, so as to mitigate their detrimental effects on future global food security and plant biodiversity.

Future Scenarios for Plant Virus Pathogens as Climate Change Progresses.

Potato virus Y (PVY, genus Potyvirus, family Potyviridae) causes high economic losses worldwide, especially in the production of seed potatoes (Solanum tuberosum). PVY control systems rely on measuring virus pressure and vector pressure in the field. Calculation of the vector pressure is based on the relative efficiency factors (REFs) of aphid species. These REFs express the transmission efficiency of aphid species in relation to the transmission efficiency of Myzus persicae, the most efficient vector of PVY. In this paper, we report on the determination of aphids' relative transmission efficiency factors (REFs) for isolates of the PVY strains PVYN, PVYNTN and PVYN-Wi. Biotype Mp2 of M. persicae was tested for its transmission efficiency for six PVY isolates (one PVYN, three PVYNTN and two PVYN-Wi isolates) and showed comparable average transmission efficiencies for all isolates. The transmission rate of this biotype for the six PVY isolates was set to 1 and Mp2 was used as an internal control in transmission experiments to determine the REFs of three other biotypes of M. persicae and 16 other aphid species (three biotypes per species when available) for the six PVY isolates. Comparing the calculated REFs for PVYN with the REFs reported in the previous century for PVYN, we observe overall comparable REFs, except for Aphis fabae, Aphis spp., Hyperomyzus lactucae, Macrosiphum euphorbiae and Rhopalosiphum padi, which have a lower REF in our experiments, and Aphis frangulae and Phorodon humuli, which have now a higher REF. Comparing the new REFs found for the PVYNTN strains with the new REFs for PVYN, we observe that they are overall comparable, except for A. frangulae (0.17 compared with 0.53) and Schizaphis graminum (0.05 compared with 0.00). Comparing the REFs calculated for PVYN-Wi with those calculated for PVYN, we can observe six aphid species with higher REFs (Acyrthosiphon pisum, A. fabae, Aphis nasturtii, Aphis spp., P. humuli and R. padi). Only the species A. frangulae shows a lower REF for PVYN-Wi compared with the transmission efficiency of PVYN. Three aphid species (Aulacorthum solani, Myzus ascalonicus and S. graminum) for which no REF was determined earlier were found to be capable to transmit PVY and their REFs were determine

Determination of aphid transmission efficiencies for N, NTN and Wilga strains of Potato virus Y

Eriophyoid mites (Acari Eriophyoidea) are phytophagous arthropods forming intimate relationships with their host plants. These mites are associated with annual and perennial plants including ferns, and are highly specialized with a dominant monophagy. They can be classified in different ecological classes, i.e., vagrant, gall-making and refuge-seeking species. Many of them are major pests and some of them are vectors of plant pathogens. This paper critically reviews the knowledge on eriophyoids of agricultural importance with emphasis on sources for host plant resistance to these mites. The role of species belonging to the family Eriophyidae as vectors of plant viruses is discussed. Eriophyoid-host plant interactions, the susceptibility within selected crops and main host plant tolerance/resistance mechanisms are discussed. Fundamental concepts, subjects, and problems emerged in this review are pointed out and studies are suggested to clarify some controversial points.

An Intimate Relationship Between Eriophyoid Mites and Their Host Plants - A Review.

Eriophyid mite-transmitted, multipartite, negative-sense RNA plant viruses with membrane-bound spherical virions are classified in the genus Emaravirus. We report here that the eriophyid mite-transmitted Wheat mosaic virus (WMoV), an Emaravirus, contains eight genomic RNA segments, the most in a known negative-sense RNA plant virus. Remarkably, two RNA 3 consensus sequences, encoding the nucleocapsid protein, were found with 12.5% sequence divergence, while no heterogeneity was observed in the consensus sequences of additional genomic RNA segments. The RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid, and P4 proteins of WMoV exhibited limited sequence homology with the orthologous proteins of other emaraviruses, while proteins encoded by additional genomic RNA segments displayed no significant homology with proteins reported in GenBank, suggesting that the genus Emaravirus evolved further with a divergent octapartite genome. Phylogenetic analyses revealed that WMoV formed an evolutionary link between members of the Emaravirus genus and the family Bunyaviridae. Furthermore, genomic-length virus- and virus-complementary (vc)-sense strands of all WMoV genomic RNAs accumulated asymmetrically in infected wheat, with 10- to 20-fold more virus-sense genomic RNAs than vc-sense RNAs. These data further confirm the octapartite negative-sense polarity of the WMoV genome. In WMoV-infected wheat, subgenomic-length mRNAs of vc sense were detected for genomic RNAs 3, 4, 7, and 8 but not for other RNA species, suggesting that the open reading frames present in the complementary sense of genomic RNAs are expressed through subgenomic- or near-genomic-length vc-sense mRNAs.

IMPORTANCE Wheat mosaic virus (WMoV), an Emaravirus, is the causal agent of High Plains disease of wheat and maize. In this study, we demonstrated that the genome of WMoV comprises eight negative-sense RNA segments with an unusual sequence polymorphism in an RNA encoding the nucleocapsid protein but not in the additional genomic RNA segments. WMoV proteins displayed weak or no homology with reported emaraviruses, suggesting that the genus Emaravirus further evolved with a divergent octapartite genome. The current study also examined the profile of WMoV RNA accumulation in wheat and provided evidence for the synthesis of subgenomic-length mRNAs of virus complementary sense. This is the first report to demonstrate that emaraviruses produce subgenomic-length mRNAs that are most likely utilized for genome expression. Importantly, this study facilitates the examination of gene functions and virus diversity and the development of effective diagnostic methods and management strategies for an economically important but poorly understood virus.

/pdf/an-eriophyid-mite-transmitted-plant-virus-contains-eight-3rnq0fs1dt.pdf

An Eriophyid Mite-transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1443&context=plantpathpapers

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: formation of dense fluorescent aggregates for sensitive virus tracking.

Wheat is an important food grain worldwide and the primary dryland crop in the western Great Plains. A complex of three wheat curl mite (WCM)-transmitted viruses (Wheat streak mosaic virus, High plains virus, and Triticum mosaic virus [TriMV]) is a cause of serious loss in winter wheat production in the Great Plains. TriMV was first reported in Kansas in 2006 and later found in most other Great Plains states. Currently, three populations of WCM have been identified by genetic characterization and differential responses to mite resistance genes in wheat. In this study, we examined TriMV transmission by these three WCM populations: 'Nebraska' (NE), 'Montana' (MT), and 'South Dakota' (SD). Mite transmission using single-mite transfers revealed that the NE WCM population transmitted TriMV at 41%, while the MT and SD WCM populations failed to transmit TriMV. In multi-mite transfers, the NE WCM population transmitted TriMV at 100% level compared with 2.5% transmission by MT and SD WCM populations. Interestingly, NE mites transferred during the quiescent stages following the first and second instar transmitted TriMV at a 39 to 40% rate, suggesting that immature mites were able to acquire the virus and maintain it through molting. In addition, mite survival for single-mite transfers was significantly lower for NE mites when transferred from TriMV-inoculated source plants (60%) compared with mock-inoculated source plants (84%). This demonstrates potentially negative effects on WCM survival from TriMV. TriMV transmission differences demonstrated in this study underscore the importance of identification of mite genotypes for future studies with TriMV.

/pdf/differential-transmission-of-triticum-mosaic-virus-by-wheat-1pysspo09z.pdf

Differential Transmission of Triticum mosaic virus by Wheat Curl Mite Populations Collected in the Great Plains

Wheat streak mosaic virus (WSMV), type member of the genus Tritimovirus in the family Potyviridae, is an economically important virus causing annual average yield losses of approximately 2 to 3% in winter wheat across the Great Plains. The wheat curl mite (WCM), Aceria tosichella, transmits WSMV along with two other viruses found throughout the Great Plains of the United States. Two common genotypes of WSMV (Sidney 81 and Type) in the United States share 97.6% nucleotide sequence identity but their transmission relationships with the WCM are unknown. The objective of this study was to determine transmission of these two isolates of WSMV by five WCM populations ('Nebraska', 'Montana', 'South Dakota', 'Type 1', and 'Type 2'). Nonviruliferous mites from each population were reared on wheat source plants mechanically inoculated with either Sidney 81 or Type WSMV isolates. For each source plant, individual mites were transferred to 10 separate test plants and virus transmission was determined by a double-antibody sandwich enzyme-linked immunosorbent assay. Source plants were replicated nine times for each treatment (90 individual mite transfers). Results indicate that three mite populations transmitted Sidney 81 at higher rates compared with Type. Two mite populations (Nebraska and Type 2) transmitted Sidney 81 and Type at higher rates compared with the other three populations. Results from this study demonstrate that interactions between virus isolates and mite populations influence the epidemiology of WSMV.

/pdf/differential-transmission-of-two-isolates-of-wheat-streak-3r0jm2aupd.pdf

Differential Transmission of Two Isolates of Wheat streak mosaic virus by Five Wheat Curl Mite Populations

The wheat curl mite, Aceria tosichella Keifer, is an eriophyid pest of wheat, although its primary economic impact on wheat is due to the transmission of Wheat streak mosaic (WSMV), Wheat mosaic (also known as High Plains virus), and Triticum mosaic (TriMV) viruses. These viruses cause significant annual losses in winter wheat production throughout the western Great Plains. Temperature and humidity are factors that often influence arthropod survival, especially during dispersal from their hosts, yet the impact of these two factors on off-host survival has not been documented for wheat curl mite. Pathogen-infected host plants often influence the biology and behavior of vectors, yet it is not known if virus-infected wheat affects off-host survival of wheat curl mite. The objectives of this study were to 1) determine if temperature, relative humidity, and mite genotype impact off-host survival of wheat curl mite and 2) determine the effect of WSMV- and TriMV-infected host plants on off-host survival of wheat curl mite. Temperature and relative humidity significantly affected off-host survival of wheat curl mite. Length of survival decreased with increasing temperature (106.2 h at 10°C and 17.0 h at 30°C) and decreasing relative humidity (78.1 h at 95 and 21.3 h at 2%). Mites from TriMV-infected host plants had ∼20% reduction in survival at 20°C compared with those from WSMV-infected plants. The duration of off-host survival of wheat curl mite is influenced by environmental conditions. Management strategies that target a break in host presence will greatly reduce mite densities and virus spread and need to account for these limits.

/pdf/the-effect-of-temperature-relative-humidity-and-virus-5dxaud2l7b.pdf

Anthony J. McMechan

Papers

An Eriophyid Mite-transmitted Plant Virus Contains Eight Genomic RNA Segments with Unusual Heterogeneity in the Nucleocapsid Protein

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: formation of dense fluorescent aggregates for sensitive virus tracking.

Differential Transmission of Triticum mosaic virus by Wheat Curl Mite Populations Collected in the Great Plains

Differential Transmission of Two Isolates of Wheat streak mosaic virus by Five Wheat Curl Mite Populations

The Effect of Temperature, Relative Humidity, and Virus Infection Status on off-host Survival of the Wheat Curl Mite (Acari: Eriophyidae).