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.

Horizontally transferred genes as RNA interference targets for aphid and whitefly control.

RNA interference (RNAi)-based technologies are starting to be commercialized as a new approach for agricultural pest control. Horizontally transferred genes (HTGs), which have been transferred into insect genomes from viruses, bacteria, fungi or plants, are attractive targets for RNAi-mediated pest control. HTGs are often unique to a specific insect family or even genus, making it unlikely that RNAi constructs targeting such genes will have negative effects on ladybugs, lacewings and other beneficial predatory insect species. In this study, we sequenced the genome of a red, tobacco-adapted isolate of Myzus persicae (green peach aphid) and bioinformatically identified 30 HTGs. We then used plant-mediated virus-induced gene silencing (VIGS) to show that several HTGs of bacterial and plant origin are important for aphid growth and/or survival. Silencing the expression of fungal-origin HTGs did not affect aphid survivorship but decreased aphid reproduction. Importantly, although there was uptake of plant-expressed RNA by Coccinella septempunctata (seven-spotted ladybugs) via the aphids that they consumed, we did not observe negative effects on ladybugs from aphid-targeted VIGS constructs. To demonstrate that this approach is more broadly applicable, we also targeted five Bemisia tabaci (whitefly) HTGs using VIGS and demonstrated that knockdown of some of these genes affected whitefly survival. As functional HTGs have been identified in the genomes of numerous pest species, we propose that these HTGs should be explored further as efficient and safe targets for control of insect pests using plant-mediated RNA interference.

Acylsugars protect Nicotiana benthamiana against insect herbivory and desiccation.

KEY MESSAGE: Nicotiana benthamiana acylsugar acyltransferase (ASAT) is required for protection against desiccation and insect herbivory. Knockout mutations provide a new resource for investigation of plant-aphid and plant-whitefly interactions. Nicotiana benthamiana is used extensively as a transient expression platform for functional analysis of genes from other species. Acylsugars, which are produced in the trichomes, are a hypothesized cause of the relatively high insect resistance that is observed in N. benthamiana. We characterized the N. benthamiana acylsugar profile, bioinformatically identified two acylsugar acyltransferase genes, ASAT1 and ASAT2, and used CRISPR/Cas9 mutagenesis to produce acylsugar-deficient plants for investigation of insect resistance and foliar water loss. Whereas asat1 mutations reduced accumulation, asat2 mutations caused almost complete depletion of foliar acylsucroses. Three hemipteran and three lepidopteran herbivores survived, gained weight, and/or reproduced significantly better on asat2 mutants than on wildtype N. benthamiana. Both asat1 and asat2 mutations reduced the water content and increased leaf temperature. Our results demonstrate the specific function of two ASAT proteins in N. benthamiana acylsugar biosynthesis, insect resistance, and desiccation tolerance. The improved growth of aphids and whiteflies on asat2 mutants will facilitate the use of N. benthamiana as a transient expression platform for the functional analysis of insect effectors and resistance genes from other plant species. Similarly, the absence of acylsugars in asat2 mutants will enable analysis of acylsugar biosynthesis genes from other Solanaceae by transient expression.

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.

Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus-Petunia isolate and its associated betasatellite.

BACKGROUND: Geminiviruses cause major losses to several economically important crops. Pedilanthus leaf curl virus (PeLCV) is a pathogenic geminivirus that appeared in the last decade and is continuously increasing its host range in Pakistan and India. This study reports the identification and characterization of PeLCV-Petunia from ornamental plants in Pakistan, as well as geographical, phylogenetic, and recombination analysis. METHODS: Viral genomes and associated satellites were amplified, cloned, and sequenced from Petunia atkinsiana plants showing typical geminivirus infection symptoms. Virus-satellite complex was analyzed for phylogenetic and recombination pattern. Infectious clones of isolated virus and satellite molecules were constructed using a partial dimer strategy. Infectivity analysis of PeLCV alone and in combination with Digera yellow vein betasatellite (DiYVB) was performed by Agrobacterium infiltration of Nicotiana benthamiana and Petunia atkinsiana plants with infectious clones. RESULTS: PeLCV, in association with DiYVB, was identified as the cause of leaf curl disease on P. atkinsiana plants. Sequence analysis showed that the isolated PeLCV is 96-98% identical to PeLCV from soybean, and DiYVB has 91% identity to a betasatellite identified from rose. Infectivity analysis of PeLCV alone and in combination with DiYVB, performed by Agrobacterium infiltration of infectious clones in N. benthamiana and P. atkinsiana plants, resulted in mild and severe disease symptoms 14 days after infiltration, respectively, demonstrating that these viruses are natural disease-causing agents. Southern blot hybridization indicated successful replication of the virus-betasatellite complex in the infected plants. Phylogenetic analysis suggests that PeLCV originated from Pakistan and later spread to India. Recombination analysis predicted that PeLCV is a donor parent for recombination and evolution of two important begomoviruses, Papaya leaf curl virus (PaLCuV) and Radish leaf curl virus (RaLCuV). The molecular phylogeny of genes encoding coat protein (CP) and replication associated protein (Rep) depict a complex evolutionary pattern of the viruses, with wide diversity in both of the genes. CONCLUSIONS: This study presents PeLCV and DiYVB as a new natural combination resulting in leaf curl disease on P. atkinsiana plants. Phylogenetic analysis, in addition to recent agricultural reports, identify PeLCV as an emerging broad host range Begomovirus that is resident in Pakistan and, more recently, has also spread to India. Recombination analysis showed that PeLCV was involved in a natural recombinational event leading to the evolution of two recombinant begomoviruses, RaLCuV and PaLCuV.

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.

REPercussions: how geminiviruses recruit host factors for replication.

Circular single-stranded DNA viruses of the family Geminiviridae encode replication-associated protein (Rep), which is a multifunctional protein involved in virus DNA replication, transcription of virus genes, and suppression of host defense responses. Geminivirus genomes are replicated through the interaction between virus Rep and several host proteins. The Rep also interacts with itself and the virus replication enhancer protein (REn), which is another essential component of the geminivirus replicase complex that interacts with host DNA polymerases α and δ. Recent studies revealed the structural and functional complexities of geminivirus Rep, which is believed to have evolved from plasmids containing a signature domain (HUH) for single-stranded DNA binding with nuclease activity. The Rep coding sequence encompasses the entire coding sequence for AC4, which is intricately embedded within it, and performs several overlapping functions like Rep, supporting virus infection. This review investigated the structural and functional diversity of the geminivirus Rep.

Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus.

The replication-associated (Rep) proteins of pathogenic begomoviruses, including cotton leaf curl Multan virus (CLCuMuV) and pedilanthus leaf curl virus (PeLCV), interact with the DNA replication machinery of their eukaryotic hosts. The analysis of Rep protein sequences showed that there is 13-28% sequence variation among CLCuMuV and PeLCV isolates, with phylogenetic clusters that can separated at least in part based on the country of origin of the respective viruses. To identify specific host factors involved in the virus replication cycle, we conducted yeast two-hybrid assays to detect possible interactions between the CLCuMuV and PeLCV Rep proteins and 30 protein components of the Saccharomyces cerevisiae DNA replication machinery. This showed that the proliferating cell nuclear antigen (PCNA) protein of S. cerevisiae interacts with Rep proteins from both CLCuMuV and PeLCV. We used the yeast PCNA sequence in BLAST comparisons to identify two PCNA orthologs each in Gossypium hirsutum (cotton), Arabidopsis thaliana (Arabidopsis), and Nicotiana benthamiana (tobacco). Sequence comparisons showed 38-40% identity between the yeast and plant PCNA proteins, and > 91% identity among the plant PCNA proteins, which clustered together in one phylogenetic group. The expression of the six plant PCNA proteins in the yeast two-hybrid system confirmed interactions with the CLCuMuV and PeLCV Rep proteins. Our results demonstrate that the interaction of begomovirus Rep proteins with eukaryotic PCNA proteins is strongly conserved, despite significant evolutionary variation in the protein sequences of both of the interacting partners.

Novel Ampeloviruses Infecting Cassava in Central Africa and the South-West Indian Ocean Islands.

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified.

Genome sequence of the corn leaf aphid (Rhopalosiphum maidis Fitch).

BACKGROUND: The corn leaf aphid (Rhopalosiphum maidis Fitch) is the most economically damaging aphid pest on maize (Zea mays), one of the world's most important grain crops. In addition to causing direct damage by removing photoassimilates, R. maidis transmits several destructive maize viruses, including maize yellow dwarf virus, barley yellow dwarf virus, sugarcane mosaic virus, and cucumber mosaic virus. FINDINGS: The genome of a parthenogenetically reproducing R. maidis clone was assembled with a combination of Pacific Biosciences (207-fold coverage) and Illumina (83-fold coverage) sequencing. The 689 assembled contigs, which have an N50 size of 9.0 megabases (Mb) and a low level of heterozygosity, were clustered using Phase Genomics Hi-C interaction maps. Consistent with the commonly observed 2n = 8 karyotype of R. maidis, most of the contigs (473 spanning 321 Mb) were successfully oriented into 4 scaffolds. The genome assembly captured the full length of 95.8% of the core eukaryotic genes, indicating that it is highly complete. Repetitive sequences accounted for 21.2% of the assembly, and a total of 17,629 protein-coding genes were predicted with integrated evidence from ab initio and homology-based gene predictions and transcriptome sequences generated with both Pacific Biosciences and Illumina. An analysis of likely horizontally transferred genes identified 2 from bacteria, 7 from fungi, 2 from protozoa, and 9 from algae. Repeat elements, transposons, and genes encoding likely detoxification enzymes (cytochrome P450s, glutathione S-transferases, carboxylesterases, uridine diphosphate-glucosyltransferases, and ABC transporters) were identified in the genome sequence. Other than Buchnera aphidicola (642,929 base pairs, 602 genes), no endosymbiont bacteria were found in R. maidis. CONCLUSIONS: A high-quality R. maidis genome was assembled at the chromosome level. This genome sequence will enable further research related to ecological interactions, virus transmission, pesticide resistance, and other aspects of R. maidis biology. It also serves as a valuable resource for comparative investigation of other aphid species.

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.

Applications of New Breeding Technologies for Potato Improvement.

The first decade of genetic engineering primarily focused on quantitative crop improvement. With the advances in technology, the focus of agricultural biotechnology has shifted toward both quantitative and qualitative crop improvement, to deal with the challenges of food security and nutrition. Potato (Solanum tuberosum L.) is a solanaceous food crop having potential to feed the populating world. It can provide more carbohydrates, proteins, minerals, and vitamins per unit area of land as compared to other potential food crops, and is the major staple food in many developing countries. These aspects have driven the scientific attention to engineer potato for nutrition improvement, keeping the yield unaffected. Several studies have shown the improved nutritional value of potato tubers, for example by enhancing Amaranth Albumin-1 seed protein content, vitamin C content, ß-carotene level, triacylglycerol, tuber methionine content, and amylose content, etc. Removal of anti-nutritional compounds like steroidal glycoalkaloids, acrylamide and food toxins is another research priority for scientists and breeders to improve potato tuber quality. Trait improvement using genetic engineering mostly involved the generation of transgenic products. The commercialization of these engineered products has been a challenge due to consumer preference and regulatory/ethical restrictions. In this context, new breeding technolgies like TALEN (transcription activator-like effector nucleases) and CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated 9) have been employed to generate transgene-free products in a more precise, prompt and effective way. Moreover, the availability of potato genome sequence and efficient potato transformation systems have remarkably facilitated potato genetic engineering. Here we summarize the potato trait improvement and potential application of new breeding technologies (NBTs) to genetically improve the overall agronomic profile of potato.

An Insight into Cotton Leaf Curl Multan Betasatellite, the Most Important Component of Cotton Leaf Curl Disease Complex.

Cotton leaf curl disease (CLCuD) is one of the most economically important diseases and is a constraint to cotton production in major producers, Pakistan and India. CLCuD is caused by monopartite plant viruses belonging to the family Geminiviridae (genus Begomovirus), in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMuB) belonging to a newly-established family Tolecusatellitidae (genus Betasatellite). CLCuMuB has a small genome (ca. 1350 nt) with a satellite conserved region, an adenine-rich region and a single gene that encodes for a multifunctional ßC1 protein. CLCuMuB ßC1 protein has a major role in pathogenicity and symptom determination, and alters several host cellular functions like autophagy, ubiquitination, and suppression of gene silencing, to assist CLCuD infectivity. Efficient trans-replication ability of CLCuMuB with several monopartite and bipartite begomoviruses, is also associated with the rapid evolution and spread of CLCuMuB. In this article we comprehensively reviewed the role of CLCuMuB in CLCuD, focusing on the ßC1 functions and its interactions with host proteins.

Multiple begomoviruses found associated with cotton leaf curl disease in Pakistan in early 1990 are back in cultivated cotton.

The first epidemic of cotton leaf curl disease (CLCuD) in early 1990's in the Indian subcontinent was associated with several distinct begomoviruses along with a disease-specific betasatellite. Resistant cotton varieties were introduced in late 1990's but soon resistance was broken and was associated with a single recombinant begomovirus named Burewala strain of Cotton leaf curl Kokhran virus that lacks a full complement of a gene encoding a transcription activator protein (TrAP). In order to understand the ongoing changes in CLCuD complex in Pakistan, CLCuD affected plants from cotton fields at Vehari were collected. Illumina sequencing was used to assess the diversity of CLCuD complex. At least three distinct begomoviruses characterized from the first epidemic; Cotton leaf curl Multan virus, Cotton leaf curl Kokhran virus and Cotton leaf curl Alabad virus, several distinct species of alphasatellites and cotton leaf curl Multan betasatellite were found associated with CLCuD. These viruses were also cloned and sequenced through Sanger sequencing to confirm the identity of the begomoviruses and that all clones possessed a full complement of the TrAP gene. A new strain of betasatellite was identified here and named CLCuMuBVeh. The implications of these findings in efforts to control CLCuD are discussed.

Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses.

The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent.