Jonathan Shao

Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII)

Phytoplasmas, the causal agents of numerous plant diseases, are insect-vector-transmitted, cell-wall-less bacteria descended from ancestral low-G+C-content Gram-positive bacteria in the Bacillus–Clostridium group. Despite their monophyletic origin, widely divergent phytoplasma lineages have evolved in adaptation to specific ecological niches. Classification and taxonomic assignment of phytoplasmas have been based primarily on molecular analysis of 16S rRNA gene sequences because of the inaccessibility of measurable phenotypic characters suitable for conventional microbial characterization. In the present study, an interactive online tool, iPhyClassifier, was developed to expand the efficacy and capacity of the current 16S rRNA gene sequence-based phytoplasma classification system. iPhyClassifier performs sequence similarity analysis, simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) profiles. Based on calculated RFLP pattern similarity coefficients and overall sequence similarity scores, iPhyClassifier makes instant suggestions on tentative phytoplasma 16Sr group/subgroup classification status and ‘Candidatus Phytoplasma’ species assignment. Using iPhyClassifier, we revised and updated the classification of strains affiliated with the peach X-disease phytoplasma group. The online tool can be accessed at http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier.html.

Analysis of the Alfalfa Root Transcriptome in Response to Salinity Stress

Salinity is one of the major abiotic factors affecting alfalfa productivity. Identifying genes that control this complex trait will provide critical insights for alfalfa breeding programs. To date, no studies have been published on a deep sequencing-based profiling of the alfalfa transcriptome in response to salinity stress. Observations gathered through research on reference genomes may not always be applicable to alfalfa. In this work, Illumina RNA-sequencing was performed in two alfalfa genotypes contrasting in salt tolerance, in order to estimate a broad spectrum of genes affected by salt stress. A total of 367,619,586 short reads were generated from cDNA libraries originated from roots of both lines. More than 60,000 tentative consensus sequences (TCs) were obtained and, among them, 74.5% had a significant similarity to proteins in the NCBI database. Mining of simple sequence repeats (SSRs) from all TCs revealed 6,496 SSRs belonging to 3,183 annotated unigenes. Bioinformatics analysis showed that the expression of 1,165 genes, including 86 transcription factors (TFs), was significantly altered under salt stress. About 40% of differentially expressed genes were assigned to known gene ontology (GO) categories using Arabidopsis GO. A random check of differentially expressed genes by quantitative real-time PCR confirmed the bioinformatic analysis of the RNA-seq data. A number of salt-responsive genes in both tested genotypes were identified and assigned to functional classes, and gene candidates with roles in the adaptation to salinity were proposed. Alfalfa-specific data on salt-responsive genes obtained in this work will be useful in understanding the molecular mechanisms of salinity tolerance in alfalfa.

Genome and secretome analysis of the hemibiotrophic fungal pathogen, Moniliophthora roreri, which causes frosty pod rot disease of cacao: mechanisms of the biotrophic and necrotrophic phases.

BackgroundThe basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp.ResultsWe sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase.ConclusionsGenome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease.

A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings. A library of small RNAs was constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8,717 nucleotides [nt]) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains: namely, methyltransferase (MTR), cysteine protease-like, FtsJ-MTR, helicase (Hel), and RNA-dependent RNA polymerase (RdRp); whereas ORF2 encoded the putative coat protein. RNA2 (4,989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24, and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3 untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58 and 50% nucleotide identities, respectively, with known CiLV-C sequences and, thus, it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp, and MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome-specific RT-PCR primers was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

Transcriptome analysis of resistant and susceptible alfalfa cultivars infected with root-knot nematode Meloidogyne incognita.

Nematodes are one of the major limiting factors in alfalfa production. Root-knot nematodes (RKN, Meloidogyne spp.) are widely distributed and economically important sedentary endoparasites of agricultural crops and they may inflict significant damage to alfalfa fields. As of today, no studies have been published on global gene expression profiling in alfalfa infected with RKN or any other plant parasitic nematode. Very little information is available about molecular mechanisms that contribute to pathogenesis and defense responses in alfalfa against these pests and specifically against RKN. In this work, we performed root transcriptome analysis of resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa cultivars infected with RKN Meloidogyne incognita, widespread root-knot nematode species and a major pest worldwide. A total of 1,701,622,580 pair-end reads were generated on an Illumina Hi-Seq 2000 platform from the roots of both cultivars and assembled into 45,595 and 47,590 transcripts in cvs Moapa 69 and Lahontan, respectively. Bioinformatic analysis revealed a number of common and unique genes that were differentially expressed in susceptible and resistant lines as a result of nematode infection. Although the susceptible cultivar showed a more pronounced defense response to the infection, feeding sites were successfully established in its roots. Characteristically, basal gene expression levels under normal conditions differed between the two cultivars as well, which may confer advantage to one of the genotypes toward resistance to nematodes. Differentially expressed genes were subsequently assigned to known Gene Ontology categories to predict their functional roles and associated biological processes. Real-time PCR validated expression changes in genes arbitrarily selected for experimental confirmation. Candidate genes that contribute to protection against M. incognita in alfalfa were proposed and alfalfa-nematode interactions with respect to resistance are discussed.

Genome Assembly of Citrus Leprosis Virus Nuclear Type Reveals a Close Association with Orchid Fleck Virus

ABSTRACT The complete genome of citrus leprosis virus nuclear type (CiLV-N) was identified by small RNA sequencing utilizing leprosis-affected citrus samples collected from the state of Querétaro, Mexico. The nucleotide identity and phylogenetic analysis indicate that CiLV-N is very closely related to orchid fleck virus, which typically infects Cymbidium species.

The iPhyClassifier, an Interactive Online Tool for Phytoplasma Classification and Taxonomic Assignment

The iPhyClassifier is an internet-based research tool for quick identification and classification of diverse phytoplasmas. The iPhyClassifier simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) patterns. Based on RFLP pattern similarity coefficient scores, the iPhyClassifier gives instant suggestions on group and subgroup classification status of the phytoplasma strains under study. The iPhyClassifier also aligns the query sequences with that of reference strains of all previously described Candidatus Phytoplasma species, -calculates sequence similarity scores, and assigns the phytoplasmas under study into respective Ca. Phytoplasma species as related strains according to the guidelines set forth by the Phytoplasma Taxonomy Group of the International Research Program on Comparative Mycoplasmology. Additional functions of the iPhyClassifier include delineation of potentially new phytoplasma groups and subgroups as well as new Ca. Phytoplasma species. This chapter describes the program components, the operational procedure, and the underlying principles of the iPhyClassifier operation. The chapter also provides hints on how to interpret the results.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5 untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

A Case Study on Discovery of Novel Citrus Leprosis Virus Cytoplasmic Type 2 Utilizing Small RNA Libraries by Next Generation Sequencing and Bioinformatic Analyses

The advent of innovative sequencing technology referred to as “Next-Generation” Sequencing (NGS), provides a new approach to identify the ‘unknown known’ and ‘unknown unknown’ viral pathogens without a priori knowledge. The genomes of plant viruses can be rapidly determined even when occurring at extremely low titers in the infected host. The method is based on massively parallel sequencing of the population of small RNA molecules 18-35 nucleotides in length produced by RNA silencing host defense. Improvements in chemistries, bioinformatic tools and advances in engineering has reduced the costs of NGS, increased its accessibility, and enabled its application in the field of plant virology. In this review, we discuss the utilization of the Illumina GA IIX platform combined with the application of molecular biology and bioinformatic tools for the discovery of a novel cytoplasmic Citrus leprosis virus (CiLV). This new virus produced symptoms typical of CiLV but was not detected with either serological or PCR-based assays for the previously described virus. The new viral genome was also present in low titer in sweet orange (Citrus sinensis), an important horticultural crop with incomplete genomic resources. This is a common situation in horticultural research and provides an example of the broader utility of this approach. In addition to the discovery of novel viruses, the sequence data may be useful for studies of viral evolution and ecology and the interactions between viral and host transcriptomes.