Harnessing plant viruses in the metagenomics era: from the development of infectious clones to applications.

Recent metagenomic studies which focused on virus characterization in the entire plant environment have revealed a remarkable viral diversity in plants. The exponential discovery of viruses also requires the concomitant implementation of high-throughput methods to perform their functional characterization. Despite several limitations, the development of viral infectious clones remains a method of choice to understand virus biology, their role in the phytobiome, and plant resilience. Here, we review the latest approaches for efficient characterization of plant viruses and technical advances built on high-throughput sequencing and synthetic biology to streamline assembly of viral infectious clones. We then discuss the applications of plant viral vectors in fundamental and applied plant research as well as their technical and regulatory limitations, and we propose strategies for their safer field applications.

Analysis of a tetraploid cotton line Mac7 transcriptome reveals mechanisms underlying resistance against the whitefly Bemisia tabaci.

Whitefly inflicts both direct and indirect losses to cotton crop. Whitefly resistant cotton germplasm is a high priority and considered among the best possible solutions to mitigate this issue. In this study, we evaluated cotton leaf curl disease (CLCuD) resistant cotton line Mac7 under whitefly stress. Furthermore, we utilized the already available transcriptome data of Mac7 concerning whitefly stress to elucidate associated mechanisms and identify functionally important genes in cotton. In transcriptomic data analysis, differentially expressed genes (DEGs) were found involved in complex relay pathways, activated on whitefly exposure. The response implicates signalling through resistance genes (R-genes), MAPK, ROS, VQs or RLKs, transcription factors, which leads to the activation of defence responses including, Ca2+messengers, phytohormonal cross-talk, gossypol, flavonoids, PhasiRNA and susceptibility genes (S-genes). The qRT-PCR assay of 10 functionally important genes also showed their involvement in differential responses at 24 and 48 h post whitefly infestation. Briefly, our study helps in understanding the resistant nature of Mac7 under whitefly stress.

Circular DNA enrichment sequencing reveals the viral/satellites genetic diversity associated with the third epidemic of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is the most important limiting factor for cotton production in Pakistan. The CLCuD passed through two major epidemics in this region with distinct begomoviruses/satellites complexes. Since 2015 the disease has again started to appear in epidemic form, causing heavy losses to cotton crop, which we termed as the "third epidemic". We applied CIDER-seq (Circular DNA Enrichment Sequencing), a recently developed sequencing method for PCR-free virus enrichment to produce a full length read of a single circular viral genome coupled with Sanger sequencing to explore the genetic diversity of the disease complex. We identified a highly recombinant strain of Cotton leaf curl Multan virus and a recently evolved strain of Cotton leaf curl Multan betasatellite that are dominant in all major cotton growing regions in the country. Moreover, we also identified multiple species of alphasatellites with one distinct species, Mesta yellow vein mosaic alphasatellite (MeYVMA) for the first time in cotton. Relative abundance of virus and associated satellites was also determined by real-time quantitative PCR. To the best of our knowledge, this is the first study that determined the CLCuD complex associated with its third epidemic.

Plant Genetic Networks Shaping Phyllosphere Microbial Community.

Phyllosphere microbial communities inhabit the aerial plant parts, such as leaves and flowers, where they form complex molecular interactions with the host plant. Contrary to the relatively well-studied rhizosphere microbiome, scientists are just starting to understand, and potentially utilize, the phyllosphere microbiome. In this article, we summarize the recent studies that have provided novel insights into the mechanism of the host genotype shaping the phyllosphere microbiome and the possibility to select a stable and well-adapted microbiome. We also discuss the most pressing gaps in our knowledge and identify the most promising research directions and tools for understanding the assembly and function of phyllosphere microbiomes - this understanding is necessary if we are to harness phyllosphere microbiomes for improving plant growth and health in managed systems.

Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants.

To meet increasing global food demand, breeders and scientists aim to improve the yield and quality of major food crops. Plant diseases threaten food security and are expected to increase because of climate change. CRISPR genome-editing technology opens new opportunities to engineer disease resistance traits. With precise genome engineering and transgene-free applications, CRISPR is expected to resolve the major challenges to crop improvement. Here, we discuss the latest developments in CRISPR technologies for engineering resistance to viruses, bacteria, fungi, and pests. We conclude by highlighting current concerns and gaps in technology, as well as outstanding questions for future research.

Alternative Routes to Improving Photosynthesis in Field Crops.

Photosynthesis is an important biochemical reaction that forms the basis of all food chains. Its efficiency is considered a key determinant of crop productivity. Therefore, improving photosynthetic efficiency has been a focus of intensive research. Here, we highlight simple approaches, recently reported by Chen et al. and Degen et al., to increase photosynthetic efficiency in field crops.

Virus-Induced Gene Silencing (VIGS) in Cassava Using Geminivirus Agroclones.

Virus-induced gene silencing (VIGS) is an efficient, low-cost, and rapid functional validation tool for candidate genes in planta. The VIGS approach is particularly suitable to perform reverse genetics studies in crop species. Here we present a detailed method to perform VIGS in cassava, from target gene fragment to agroinoculation and VIGS quantitation.

Full-length sequencing of circular DNA viruses and extrachromosomal circular DNA using CIDER-Seq.

Circular DNA is ubiquitous in nature in the form of plasmids, circular DNA viruses, and extrachromosomal circular DNA (eccDNA) in eukaryotes. Sequencing of such molecules is essential to profiling virus distributions, discovering new viruses and understanding the roles of eccDNAs in eukaryotic cells. Circular DNA enrichment sequencing (CIDER-Seq) is a technique to enrich and accurately sequence circular DNA without the need for polymerase chain reaction amplification, cloning, and computational sequence assembly. The approach is based on randomly primed circular DNA amplification, which is followed by several enzymatic DNA repair steps and then by long-read sequencing. CIDER-Seq includes a custom data analysis package (CIDER-Seq Data Analysis Software 2) that implements the DeConcat algorithm to deconcatenate the long sequencing products of random circular DNA amplification into the intact sequences of the input circular DNA. The CIDER-Seq data analysis package can generate full-length annotated virus genomes, as well as circular DNA sequences of novel viruses. Applications of CIDER-Seq also include profiling of eccDNA molecules such as transposable elements (TEs) from biological samples. The method takes ~2 weeks to complete, depending on the computational resources available. Owing to the present constraints of long-read single-molecule sequencing, the accuracy of circular virus and eccDNA sequences generated by the CIDER-Seq method scales with sequence length, and the greatest accuracy is obtained for molecules <10 kb long.

Molecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton (Gossypium hirsutum).

Cultivated cotton (Gossypium hirsutum) is the most important fibre crop in the world. Cotton leaf curl disease (CLCuD) is the major limiting factor and a threat to textile industry in India and Pakistan. All the local cotton cultivars exhibit moderate to no resistance against CLCuD. In this study, we evaluated an exotic cotton accession Mac7 as a resistance source to CLCuD by challenging it with viruliferous whiteflies and performing qPCR to evaluate the presence/absence and relative titre of CLCuD-associated geminiviruses/betasatellites. The results indicated that replication of pathogenicity determinant betasatellite is significantly attenuated in Mac7 and probably responsible for resistance phenotype. Afterwards, to decipher the genetic basis of CLCuD resistance in Mac7, we performed RNA sequencing on CLCuD-infested Mac7 and validated RNA-Seq data with qPCR on 24 independent genes. We performed co-expression network and pathway analysis for regulation of geminivirus/betasatellite-interacting genes. We identified nine novel modules with 52 hubs of highly connected genes in network topology within the co-expression network. Analysis of these hubs indicated the differential regulation of auxin stimulus and cellular localization pathways in response to CLCuD. We also analysed the differential regulation of geminivirus/betasatellite-interacting genes in Mac7. We further performed the functional validation of selected candidate genes via virus-induced gene silencing (VIGS). Finally, we evaluated the genomic context of resistance responsive genes and found that these genes are not specific to A or D sub-genomes of G. hirsutum. These results have important implications in understanding CLCuD resistance mechanism and developing a durable resistance in cultivated cotton.

CRISPR-Based Directed Evolution for Crop Improvement.

Directed evolution involves generating diverse sequence variants of a gene of interest to produce a desirable trait under selective pressure. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) systems can be programmed to target any genomic locus and perform targeted directed evolution. Here, we discuss the opportunities and challenges of this emerging platform for targeted crop improvement.

Engineering abiotic stress tolerance via CRISPR/ Cas-mediated genome editing.

Abiotic stresses, including drought, salinity, temperature, and heavy metals, pose a major challenge for crop production and cause substantial yield reduction worldwide. Breeding tolerant cultivars against these abiotic stresses is the most sustainable and eco-friendly approach to cope with this challenge. Advances in genome editing technologies provide new opportunities for crop improvement by employing precision genome engineering for targeted crop traits. However, the selection of the candidate genes is critical for the success of achieving the desired traits. Broadly speaking, these genes could fall into two major categories, structural and regulatory genes. Structural genes encode proteins that provide stress tolerance directly, whereas regulatory genes act indirectly by controlling the expression of other genes involved in different cellular processes. Additionally, cis-regulatory sequences are also vital for achieving stress tolerance. We propose targeting of these regulatory and/or structural genes along with the cis-regulatory sequences via the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system as a robust, efficient, and practical approach for developing crop varieties resilient to climate change. We also discuss the possibility of creating novel quantitative trait loci for abiotic stress tolerance via the CRISPR/Cas-mediated targeting of promoters. It is hoped that these genome editing tools will not only make a significant contribution towards raising novel plant types having tolerance to multiple abiotic stresses but will also aid in public acceptance of these products in years to come. This article is an attempt to critically evaluate the suitability of available tools and the target genes for obtaining plants with improved tolerance to abiotic stresses.

A Simplified Method to Engineer CRISPR/Cas9-Mediated Geminivirus Resistance in Plants.

Throughout the world, geminiviruses cause devastating losses in economically important crops, including tomato, cotton, cassava, potato, chili, and cucumber; however, control mechanisms such as genetic resistance remain expensive and ineffective. CRISPR/Cas9 is an adaptive immunity mechanism used by prokaryotes to defend against invading nucleic acids of phages and plasmids. The CRISPR/Cas9 system has been harnessed for targeted genome editing in a variety of eukaryotic species, and in plants, CRISPR/Cas9 has been used to modify or introduce many traits, including virus resistance. Recently, we demonstrated that the CRISPR/Cas9 system could be used to engineer plant immunity against geminiviruses by directly targeting the viral genome for degradation. In this chapter, we describe a detailed method for engineering CRISPR/Cas9-mediated resistance against geminiviruses. This method may provide broad, durable viral resistance, as it can target conserved regions of the viral genome and can also be customized to emerging viral variants. Moreover, this method can be used in many crop species, as it requires little or no knowledge of the host plant's genome.

Non-cultivated Cotton Species (Gossypium spp.) Act as a Reservoir for Cotton Leaf Curl Begomoviruses and Associated Satellites.

A collection of cultivated and non-cultivated species of cotton (Gossypium spp.) has been maintained for the last four decades in Multan, Pakistan. This geographical location has been observed as a hotspot for the evolution of begomoviruses and satellites associated with cotton leaf curl disease (CLCuD). Recent studies showed that begomoviruses responsible for the CLCuD epidemic in the 1990s, and that almost disappeared from the CLCuD complex in 2000s, have been observed again in CLCuD-infected cotton fields. To identify host species that acted as probable reservoirs for these viruses, we characterized begomoviruses and satellites in non-cultivated cotton species G. raimondii, G. thurberi and G. mustelinum and identified several species of CLCuD associated begomoviruses and satellites. Further, phylogenetic analysis indicated that the identified begomoviruses and beta/alphasatellites are closely related to the ones associated with the most recent CLCuD complex. qPCR indicated that the comparative level of virus significantly decreased in the presence of alphasatellites. Our results indicated that non-cultivated cotton species have been continuously challenged by diverse begomoviruses and associated satellites and act as reservoirs for CLCuD associated begomoviruses. These results provide novel insights into understanding the spread of begomoviruses and associated satellites in New World cotton species introduced into the Old World.

Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses.

BACKGROUND: Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses. RESULTS: Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus. CONCLUSIONS: Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.

A CRISPR Way for Fast-Forward Crop Domestication.

Precision crop breeding, using genome editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR) systems to improve useful traits in crop plants, holds great potential for the future of agriculture. Using CRISPR-Cas9, recent studies have engineered domestication traits in wild-relative species of tomato crop for higher nutritive value and better adaptation to diverse stresses.

Transcriptomic analysis of cultivated cotton Gossypium hirsutum provides insights into host responses upon whitefly-mediated transmission of cotton leaf curl disease.

Cotton is a commercial and economically important crop that generates billions of dollars in annual revenue worldwide. However, cotton yield is affected by a sap-sucking insect Bemisia tabaci (whitefly), and whitefly-borne cotton leaf curl disease (CLCuD). The causative agent of devastating CLCuD is led by the viruses belonging to the genus Begomovirus (family Geminiviridae), collectively called cotton leaf curl viruses. Unfortunately, the extensively cultivated cotton (Gossypium hirsutum) species are highly susceptible and vulnerable to CLCuD. Yet, the concomitant influence of whitefly and CLCuD on the susceptible G. hirsutum transcriptome has not been interpreted. In the present study we have employed an RNA Sequencing (RNA-Seq) transcriptomics approach to explore the differential gene expression in susceptible G. hirsutum variety upon infection with viruliferous whiteflies. Comparative RNA-Seq of control and CLCuD infected plants was done using Illumina HiSeq 2500. This study yielded 468 differentially expressed genes (DEGs). Among them, we identified 220 up and 248 downregulated DEGs involved in disease responses and pathogen defense. We selected ten genes for downstream RT-qPCR analyses on two cultivars, Karishma and MNH 786 that are susceptible to CLCuD. We observed a similar expression pattern of these genes in both susceptible cultivars that was also consistent with our transcriptome data further implying a wider application of our global transcription study on host susceptibility to CLCuD. We next performed weighted gene co-expression network analysis that revealed six modules. This analysis also identified highly co-expressed genes as well as 55 hub genes that co-express with ≥ 50 genes. Intriguingly, most of these hub genes are shown to be downregulated and enriched in cellular processes. Under-expression of such highly co-expressed genes suggests their roles in favoring the virus and enhancing plant susceptibility to CLCuD. We also discuss the potential mechanisms governing the establishment of disease susceptibility. Overall, our study provides a comprehensive differential gene expression analysis of G. hirsutum under whitefly-mediated CLCuD infection. This vital study will advance the understanding of simultaneous effect of whitefly and virus on their host and aid in identifying important G. hirsutum genes which intricate in its susceptibility to CLCuD.