CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

Genome sequence of the English grain aphid, Sitobion avenae and its endosymbiont Buchnera aphidicola

The English grain aphid, Sitobion avenae, is a major agricultural pest of wheat, barley and oats, and one of the principal vectors of Barley Yellow Dwarf Virus (BYDV) leading to significant reductions in grain yield, annually. Emerging resistance to and increasing regulation of insecticides has resulted in limited options for their control. Using PacBio HiFi data, we have produced a high quality draft assembly of the S. avenae genome; generating a primary assembly with a total assembly size of 475.7 Mb, and an alternate assembly with a total assembly size of 430.8 Mb. Our primary assembly was highly contiguous with only 326 contigs and a contig N50 of 15.95 Mb. Assembly completeness was estimated at 97.7% using BUSCO analysis and 31,007 and 29,037 protein coding genes were predicted from the primary and alternate assemblies, respectively. This assembly, which is to our knowledge the first for an insecticide resistant clonal lineage of English grain aphid, will provide novel insight into the molecular and mechanistic determinants of resistance and will facilitate future research into mechanisms of viral transmission and aphid behavior.

Metagenomics Analysis of the Wheat Virome Identifies Novel Plant and Fungal-Associated Viral Sequences

Wheat viruses including wheat streak mosaic virus, Triticum mosaic virus, and barley yellow dwarf virus cost substantial losses in crop yields every year. Although there have been extensive studies conducted on these known wheat viruses, currently, there is limited knowledge about all components of the wheat (Triticum aestivum L.) virome. Here, we determined the composition of the wheat virome through total RNA deep sequencing of field-collected leaf samples. Sequences were de novo assembled after removing the host reads, and BLASTx searches were conducted. In addition to the documented wheat viruses, novel plant and fungal-associated viral sequences were identified. We obtained the full genome sequence of the first umbra-like associated RNA virus tentatively named wheat umbra-like virus in cereals. Moreover, a novel bi-segmented putative virus tentatively named wheat-associated vipovirus sharing low but significant similarity with both plant and fungal-associated viruses was identified. Additionally, a new putative fungal-associated tobamo-like virus and novel putative Mitovirus were discovered in wheat samples. The discovery and characterization of novel viral sequences associated with wheat is important to determine if these putative viruses may pose a threat to the wheat industry or have the potential to be used as new biological control agents for wheat pathogens either as wild-type or recombinant viruses.

Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing

Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the Geminiviridae family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic GFP gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of Tobacco yellow dwarf virus (TYDV, genus Mastrevirus). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus Mastrevirus.

Advances in understanding the biology and epidemiology of barley yellow dwarf virus (BYDV)

A tri-trophic network of domesticated grasses (host), various aphids (vector) and barley yellow dwarf virus (pathogen) species has been spread by humans from Eurasia to the rest of the world. Understanding how climate, natural and agricultural landscapes challenge pathogens, vectors, and their natural enemies and shape their dynamics is the key to managing this pathosystem. This chapter provides an overview of this complex system and its evolution. The chapter includes a case study of biological control of aphids causing wheat BYDV in Brazil. The current challenge is to create tools that integrate knowledge of this complex pathosystem and facilitate monitoring and decision making for rational management to reduce the burden of disease.

Resistance breeding in barley against Barley yellow dwarf virus (BYDV): avoiding negative impacts on anatomy and physiology

Barley yellow dwarf (BYD) is one of the most widespread and damaging viral diseases of grasses and cereal crops worldwide. Due to an increasing risk of food losses e.g. in barley by Barley yellow dwarf virus (BYDV) as a consequence of climate change, associated by a strong demand to decrease the use of chemical insecticides, breeding for BYDV resistance is of prime importance today. This chapter describes the negative impact of BYDV on barley on multiple levels (anatomy, physiology and agronomic traits). It also demonstrates the benefits of BYDV resistance regarding a reduction in yield losses but also a decreased spread of BYDV in the field due to effects on the tritrophic interaction of virus, vector and plant. Until now, several genes and QTL are known that mediate tolerance or resistance against BYDV, respectively. The combination of genomic tools and phenotyping is the basis for the identification of these genes and recent developments facilitate to enhance this process.

Virus Diseases of Cereal and Oilseed Crops in Australia: Current Position and Future Challenges

This review summarizes research on virus diseases of cereals and oilseeds in Australia since the 1950s. All viruses known to infect the diverse range of cereal and oilseed crops grown in the continent’s temperate, Mediterranean, subtropical and tropical cropping regions are included. Viruses that occur commonly and have potential to cause the greatest seed yield and quality losses are described in detail, focusing on their biology, epidemiology and management. These are: barley yellow dwarf virus, cereal yellow dwarf virus and wheat streak mosaic virus in wheat, barley, oats, triticale and rye; Johnsongrass mosaic virus in sorghum, maize, sweet corn and pearl millet; turnip yellows virus and turnip mosaic virus in canola and Indian mustard; tobacco streak virus in sunflower; and cotton bunchy top virus in cotton. The currently less important viruses covered number nine infecting nine cereal crops and 14 infecting eight oilseed crops (none recorded for rice or linseed). Brief background information on the scope of the Australian cereal and oilseed industries, virus epidemiology and management and yield loss quantification is provided. Major future threats to managing virus diseases effectively include damaging viruses and virus vector species spreading from elsewhere, the increasing spectrum of insecticide resistance in insect and mite vectors, resistance-breaking virus strains, changes in epidemiology, virus and vectors impacts arising from climate instability and extreme weather events, and insufficient industry awareness of virus diseases. The pressing need for more resources to focus on addressing these threats is emphasized and recommendations over future research priorities provided.

A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.