Inhibition of a conserved bacterial dual-specificity phosphatase confers plant tolerance to Candidatus Liberibacter spp.

"Candidatus Liberibacter spp." are insect-vectored, fastidious, and vascular-limited phytopathogens. They are the presumptive causal agents of potato zebra chip, tomato vein clearing, and the devastating citrus greening disease worldwide. There is an urgent need to develop new strategies to control them. In this study, we characterized a dual-specificity serine/tyrosine phosphatase (STP) that is well conserved among thirty-three geographically diverse "Candidatus Liberibacter spp." and strains that infect multiple Solanaceaea and citrus spp. The STP is expressed in infected plant tissues, localized at the plant cytosol and plasma membrane, and interferes with plant cell death responses. We employed an in silico target-based molecular modeling and ligand screen to identify two small molecules with high binding affinity to STP. Efficacy studies demonstrated that the two molecules can inhibit "Candidatus Liberibacter spp." but not unrelated pathogens and confer plant disease tolerance. The inhibitors and strategies are promising means to control "Candidatus Liberibacter spp."

Xylanase Inhibitors: Defense Players in Plant Immunity with Implications in Agro-Industrial Processing.

Xylanase inhibitors (XIs) are plant cell wall proteins largely distributed in monocots that inhibit the hemicellulose degrading activity of microbial xylanases. XIs have been classified into three classes with different structures and inhibition specificities, namely Triticum aestivum xylanase inhibitors (TAXI), xylanase inhibitor proteins (XIP), and thaumatin-like xylanase inhibitors (TLXI). Their involvement in plant defense has been established by several reports. Additionally, these inhibitors have considerable economic relevance because they interfere with the activity of xylanases applied in several agro-industrial processes. Previous reviews highlighted the structural and biochemical properties of XIs and hypothesized their role in plant defense. Here, we aimed to update the information on the genomic organization of XI encoding genes, the inhibition properties of XIs against microbial xylanases, and the structural properties of xylanase-XI interaction. We also deepened the knowledge of XI regulation mechanisms in planta and their involvement in plant defense. Finally, we reported the recently studied strategies to reduce the negative impact of XIs in agro-industrial processes and mentioned their allergenicity potential.

HTSQualC is a flexible and one-step quality control software for high-throughput sequencing data analysis.

Use of high-throughput sequencing (HTS) has become indispensable in life science research. Raw HTS data contains several sequencing artifacts, and as a first step it is imperative to remove the artifacts for reliable downstream bioinformatics analysis. Although there are multiple stand-alone tools available that can perform the various quality control steps separately, availability of an integrated tool that can allow one-step, automated quality control analysis of HTS datasets will significantly enhance handling large number of samples parallelly. Here, we developed HTSQualC, a stand-alone, flexible, and easy-to-use software for one-step quality control analysis of raw HTS data. HTSQualC can evaluate HTS data quality and perform filtering and trimming analysis in a single run. We evaluated the performance of HTSQualC for conducting batch analysis of HTS datasets with 322 samples with an average ~ 1 M (paired end) sequence reads per sample. HTSQualC accomplished the QC analysis in ~ 3 h in distributed mode and ~ 31 h in shared mode, thus underscoring its utility and robust performance. In addition to command-line execution, we integrated HTSQualC into the free, open-source, CyVerse cyberinfrastructure resource as a GUI interface, for wider access to experimental biologists who have limited computational resources and/or programming abilities.

Brachypodium Phenylalanine Ammonia Lyase (PAL) Promotes Antiviral Defenses against Panicum mosaic virus and Its Satellites.

Brachypodium distachyon has recently emerged as a premier model plant for monocot biology, akin to Arabidopsis thaliana We previously reported genome-wide transcriptomic and alternative splicing changes occurring in Brachypodium during compatible infections with Panicum mosaic virus (PMV) and its satellite virus (SPMV). Here, we dissected the role of Brachypodium phenylalanine ammonia lyase 1 (PAL1), a key enzyme for phenylpropanoid and salicylic acid (SA) biosynthesis and the induction of plant defenses. Targeted metabolomics profiling of PMV-infected and PMV- plus SPMV-infected (PMV/SPMV) Brachypodium plants revealed enhanced levels of multiple defense-related hormones and metabolites such as cinnamic acid, SA, and fatty acids and lignin precursors during disease progression. The virus-induced accumulation of SA and lignin was significantly suppressed upon knockdown of B. distachyonPAL1 (BdPAL1) using RNA interference (RNAi). The compromised SA accumulation in PMV/SPMV-infected BdPAL1 RNAi plants correlated with weaker induction of multiple SA-related defense gene markers (pathogenesis related 1 [PR-1], PR-3, PR-5, and WRKY75) and enhanced susceptibility to PMV/SPMV compared to that of wild-type (WT) plants. Furthermore, exogenous application of SA alleviated the PMV/SPMV necrotic disease phenotypes and delayed plant death caused by single and mixed infections. Together, our results support an antiviral role for BdPAL1 during compatible host-virus interaction, perhaps as a last resort attempt to rescue the infected plant.IMPORTANCE Although the role of plant defense mechanisms against viruses are relatively well studied in dicots and in incompatible plant-microbe interactions, studies of their roles in compatible interactions and in grasses are lagging behind. In this study, we leveraged the emerging grass model Brachypodium and genetic resources to dissect Panicum mosaic virus (PMV)- and its satellite virus (SPMV)-compatible grass-virus interactions. We found a significant role for PAL1 in the production of salicylic acid (SA) in response to PMV/SPMV infections and that SA is an essential component of the defense response preventing the plant from succumbing to viral infection. Our results suggest a convergent role for the SA defense pathway in both compatible and incompatible plant-virus interactions and underscore the utility of Brachypodium for grass-virus biology.

Phenotypic Diversity and Association Mapping of Ascorbic Acid Content in Spinach.

Ascorbic acid (AsA), or vitamin C, is an essential nutrient for humans. In plants, AsA functions as an antioxidant during normal metabolism or in response to stress. Spinach is a highly nutritious green leafy vegetable that is consumed fresh, cooked or as a part of other dishes. One current goal in spinach breeding programs is to enhance quality and nutritional content. However, little is known about the diversity of nutritional content present in spinach germplasm, especially for AsA content. In this study, a worldwide panel of 352 accessions was screened for AsA content showing that variability in spinach germplasm is high and could be utilized for cultivar improvement. In addition, a genome-wide association study for marker-trait association was performed using three models, and associated markers were searched in the genome for functional annotation analysis. The generalized linear model (GLM), the compressed mixed linear model (CMLM) based on population parameters previously determined (P3D) and the perMarker model together identified a total of 490 significant markers distributed across all six spinach chromosomes indicating the complex inheritance of the trait. The different association models identified unique and overlapping marker sets, where 27 markers were identified by all three models. Identified high AsA content accessions can be used as parental lines for trait introgression and to create segregating populations for further genetic analysis. Bioinformatic analysis indicated that identified markers can differentiate between high and low AsA content accessions and that, upon validation, these markers should be useful for breeding programs.

Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.

A major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

GenFam: A web application and database for gene family-based classification and functional enrichment analysis.

Genome-scale studies using high-throughput sequencing (HTS) technologies generate substantial lists of differentially expressed genes under different experimental conditions. These gene lists need to be further mined to narrow down biologically relevant genes and associated functions in order to guide downstream functional genetic analyses. A popular approach is to determine statistically overrepresented genes in a user-defined list through enrichment analysis tools, which rely on functional annotations of genes based on Gene Ontology (GO) terms. Here, we propose a new computational approach, GenFam, which allows annotation, classification, and enrichment of genes based on their gene family, thus simplifying identification of candidate gene families and associated genes that may be relevant to the query. GenFam and its integrated database comprises of three hundred and eighty-four unique gene families and supports gene family analyses for sixty plant genomes. Four comparative case studies with plant species belonging to different clades and families were performed using GenFam which demonstrated its robustness and comprehensiveness over preexisting functional enrichment tools. To make it readily accessible for plant biologists, GenFam is available as a web-based application where users can input gene IDs and export enrichment results in both tabular and graphical formats. Users can also customize analysis parameters by choosing from the various statistical enrichment tests and multiple testing correction methods. Additionally, the web-based application, source code, and database are freely available to use and download. Website: http://mandadilab.webfactional.com/home/. Source code and database: http://mandadilab.webfactional.com/home/dload/.

Minor alleles are associated with white rust (Albugo occidentalis) susceptibility in spinach (Spinacia oleracea).

Minor alleles (MA) have been associated with disease incidence in human studies, enabling the identification of diagnostic risk factors for various diseases. However, allelic mapping has rarely been performed in plant systems. The goal of this study was to determine whether a difference in MA prevalence is a strong enough risk factor to indicate a likely significant difference in disease resistance against white rust (WR; Albugo occidentalis) in spinach (Spinacia oleracea). We used WR disease severity ratings (WR-DSRs) in a diversity panel of 267 spinach accessions to define resistant- and susceptibility-associated groups within the distribution scores and then tested the single-nucleotide polymorphism (SNP) variants to interrogate the MA prevalence in the most susceptible (MS) vs. most resistant (MR) individuals using permutation-based allelic association tests. A total of 448 minor alleles associated with WR severity were identified in the comparison between the 25% MS and the 25% MR accessions, while the MA were generally similar between the two halves of the interquartile range. The minor alleles in the MS group were distributed across all six chromosomes and made up ~71% of the markers that were also strongly associated with WR in parallel performed genome-wide association study. These results indicate that susceptibility may be highly determined by the disproportionate overrepresentation of minor alleles, which could be used to select for resistant plants. Furthermore, by focusing on the distribution tails, allelic mapping could be used to identify plant markers associated with quantitative traits on the most informative segments of the phenotypic distribution.

Genome-wide alternative splicing landscapes modulated by biotrophic sugarcane smut pathogen.

Alternative splicing (AS) promotes transcriptome and proteome diversity during growth, development, and stress responses in eukaryotes. Genome-wide studies of AS in sugarcane (Saccharum spp.) are lacking, mainly due to the absence of a high-quality sequenced reference genome, sugarcane's large, complex genome, and the variable chromosome numbers and polyploidy of sugarcane cultivars. Here, we analyzed changes in the sugarcane isoform-level transcriptome and AS landscape during infection with the smut fungus (Sporisorium scitamineum) using a hybrid approach involving Sorghum bicolor reference-based and Trinity de novo mapping tools. In total, this analysis detected 16,039 and 15,379 transcripts (≥2 FPKM) at 5 and 200 days after infection, respectively. A conservative estimate of isoform-level expression suggested that approximately 5,000 (14%) sugarcane genes undergo AS. Differential expression analysis of the alternatively spliced genes in healthy and smut-infected sugarcane revealed 896 AS events modulated at different stages of infection. Gene family and gene ontology functional enrichment analysis of the differentially spliced genes revealed overrepresentation of functional categories related to the cell wall, defense, and redox homeostasis pathways. Our study provides novel insight into the AS landscape of sugarcane during smut disease interactions.

An actin-depolymerizing factor from the halophyte smooth cordgrass, Spartina alterniflora (SaADF2), is superior to its rice homolog (OsADF2) in conferring drought and salt tolerance when constitutively overexpressed in rice.

Actin-depolymerizing factors (ADFs) maintain the cellular actin network dynamics by regulating severing and disassembly of actin filaments in response to environmental cues. An ADF isolated from a monocot halophyte, Spartina alterniflora (SaADF2), imparted significantly higher level of drought and salinity tolerance when expressed in rice than its rice homologue OsADF2. SaADF2 differs from OsADF2 by a few amino acid residues, including a substitution in the regulatory phosphorylation site serine-6, which accounted for its weak interaction with OsCDPK6 (calcium-dependent protein kinase), thus resulting in an increased efficacy of SaADF2 and enhanced cellular actin dynamics. SaADF2 overexpression preserved the actin filament organization better in rice protoplasts under desiccation stress. The predicted tertiary structure of SaADF2 showed a longer F-loop than OsADF2 that could have contributed to higher actin-binding affinity and rapid F-actin depolymerization in vitro by SaADF2. Rice transgenics constitutively overexpressing SaADF2 (SaADF2-OE) showed better growth, relative water content, and photosynthetic and agronomic yield under drought conditions than wild-type (WT) and OsADF2 overexpressers (OsADF2-OE). SaADF2-OE preserved intact grana structure after prolonged drought stress, whereas WT and OsADF2-OE presented highly damaged and disorganized grana stacking. The possible role of ADF2 in transactivation was hypothesized from the comparative transcriptome analyses, which showed significant differential expression of stress-related genes including interacting partners of ADF2 in overexpressers. Identification of a complex, differential interactome decorating or regulating stress-modulated cytoskeleton driven by ADF isoforms will lead us to key pathways that could be potential target for genome engineering to improve abiotic stress tolerance in agricultural crops.

Developing Growth-Associated Molecular Markers Via High-Throughput Phenotyping in Spinach.

CORE IDEAS: High-throughput imaging and genomic information can be combined to optimize marker development. Genome-wide association studies identified loci associated with plant growth traits. We identified candidate genes associated with plant growth and development. Despite advances in sequencing for genotyping, the lack of rapid, accurate, and reproducible phenotyping platforms has hampered efforts to use genetic analysis to predict traits of interest. Therefore, the use of high-throughput systems to phenotype traits related to crop growth, yield, quality, and resistance to biotic and abiotic stresses has become a major asset for breeding. Here, we assessed the efficacy of unmanned aircraft system (UAS)-based high-throughput phenotyping to obtain data for molecular marker development for spinach (Spinacia oleracea L.) improvement. We used a UAS equipped with a red-green-blue sensor to capture raw images of 284 spinach accessions throughout the crop cycle. Processed images generated orthomosaic and digital surface models for estimating canopy cover, canopy volume, and excess greenness index models. In addition, we manually recorded the number of days to bolting. Genome-wide association studies against a single-nucleotide polymorphism (SNP) panel obtained by ddRADseq identified 99 SNPs significantly associated with growth parameters. Some of these SNPs are in transcription factor and stress-response genes with possible roles in plant growth and development. The results underscore the utility of combining aerial imaging and genomic data analysis to optimize marker development. This study lays the foundation for the use of UAS-based high-throughput phenotyping for the molecular breeding of spinach.

Identification of candidate resistance genes of cotton against Aspergillus flavus infection using a comparative transcriptomics approach.

A comparative transcriptome analysis was performed using the genes significantly differentially expressed in cotton, corn and peanut in response to aflatoxin producing fungus Aspergillus flavus with an objective of identifying candidate resistance genes in cotton. Two-way analyses identified 732 unique genes to be differentially regulated by the fungus with only 26 genes common across all three crops that were considered candidate A. flavus resistance genes with an assumption that these genes have specific roles in conferring the resistance trait. Genes of membrane cellular component involved in DNA binding with involvement in defense responses were highly represented among the differentially expressed unique genes. Most (six) of these genes coded for 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily proteins. Genes encoding helix loop helix protein, alcohol dehydrogenase and UDP glycosylation transferase which were upregulated in response to both atoxigenic and toxigenic strains of A. flavus, could be potential resistance candidate genes for downstream functional manipulation to confer resistance.

Genomic Approaches to Analyze Alternative Splicing, A Key Regulator of Transcriptome and Proteome Diversity in Brachypodium distachyon.

Alternative splicing (AS) promotes transcriptome and proteome diversity in plants, which influences growth and development, and host responses to stress. Advancements in next-generation sequencing, bioinformatics, and computational biology tools have allowed biologists to investigate AS landscapes on a genome-wide scale in several plant species. Furthermore, the development of Brachypodium distachyon (Brachypodium) as a model system for grasses has facilitated comparative studies of AS within the Poaceae. These analyses revealed a plethora of genes in several biological processes that are alternatively spliced and identified conserved AS patterns among monocot and dicot plants. In this chapter, using a Brachypodium-virus pathosystem as a research template, we provide an overview of genomic and bioinformatic tools that can be used to investigate constitutive and alternative splicing in plants.

Global Awakening of Cryptic Biosynthetic Gene Clusters in Burkholderia thailandensis.

Many bacteria encode biosynthetic proteins that produce a vast array of natural products. These compounds are often synthesized during host invasion as they function as virulence factors. In addition, such secondary metabolites have yielded numerous molecular scaffolds with pharmaceutical and clinical importance. The gene clusters that encode proteins responsible for synthesis of these compounds are typically silenced or "cryptic" under laboratory growth conditions, hampering discovery of novel lead compounds. We report here that MftR is a global repressor of secondary metabolite synthesis in Burkholderia thailandensis and that urate functions as a physiologically relevant inducer of gene expression. Biosynthetic gene clusters under MftR control include those associated with production of the antimicrobial bactobolins, the iron siderophore malleobactin, and the virulence factor malleilactone. MftR also controls additional genes associated with survival in a host environment, such as genes encoding components of the type III secretion system (T3SS) and proteins linked to anaerobic respiration. This observation not only has implications for understanding activation of gene regulatory networks during host invasion, but it also paves the way for isolation of novel therapeutic leads.

Genetic Mapping of Quantitative Trait Loci for Grain Yield under Drought in Rice under Controlled Greenhouse Conditions.

Drought stress is a constant threat to rice production worldwide. Most modern rice cultivars are sensitive to drought, and the effect is severe at the reproductive stage. Conventional breeding for drought resistant (DR) rice varieties is slow and limited due to the quantitative nature of the DR traits. Identification of genes (QTLs)/markers associated with DR traits is a prerequisite for marker-assisted breeding. Grain yield is the most important trait and to this end drought yield QTLs have been identified under field conditions. The present study reports identification of drought yield QTLs under controlled conditions without confounding effects of other factors prevalent under natural conditions. A linkage map covering 1,781.5 cM with an average resolution of 9.76 cM was constructed using an F2 population from a cross between two Japonica cultivars, Cocodrie (drought sensitive) and Vandana (drought tolerant) with 213 markers distributed over 12 rice chromosomes. A subset of 59 markers (22 genic SSRs and 37 SNPs) derived from the transcriptome of the parents were also placed in the map. Single marker analysis using 187 F2 : 3 progeny identified 6 markers distributed on chromosomes 1, 5, and 8 to be associated with grain yield under drought (GYD). Composite interval mapping identified six genomic regions/quantitative trait loci (QTL) on chromosome 1, 5, 8, and 9 to be associated with GYD. QTLs located on chromosome 1 (qGYD1.2, qGYD1.3), chromosome 5 (qGYD5.1) and chromosome 8 (qGYD8.1) were contributed by Vandana alleles, whereas the QTLs, qGYD1.1 and qQYD9.1 were contributed by Cocodrie alelles. The additive positive phenotypic variance explained by the QTLs ranged from 30.0 to 34.0%. Candidate genes annotation within QTLs suggested the role of transcription factors and genes involved in osmotic potential regulation through catalytic/metabolic pathways in drought tolerance mechanism contributing to yield.

Transcriptome analysis of smooth cordgrass (Spartina alterniflora Loisel), a monocot halophyte, reveals candidate genes involved in its adaptation to salinity.

BACKGROUND: Soil salinity affects growth and yield of crop plants. Plants respond to salinity by physiological and biochemical adjustments through a coordinated regulation and expression of a cascade of genes. Recently, halophytes have attracted attention of the biologists to understand their salt adaptation mechanisms. Spartina alterniflora (smooth cordgrass) is a Louisiana native monocot halophyte that can withstand salinity up to double the strength of sea water. To dissect the molecular mechanisms underlying its salinity adaptation, leaf and root transcriptome of S. alterniflora was sequenced using 454/GS-FLX. RESULTS: Altogether, 770,690 high quality reads with an average length 324-bp were assembled de novo into 73,131 contigs (average 577-bp long) with 5.9X sequence coverage. Most unigenes (95 %) annotated to proteins with known functions, and had more than 90 % similarity to rice genes. About 28 % unigenes were considered specific to S. alterniflora. Digital expression profiles revealed significant enrichment (P < 0.01) of transporters, vacuolar proton pump members and transcription factors under salt stress, which suggested the role of ion homeostasis and transcriptional regulation in the salinity adaptation of this grass. Also, 10,805 SSRs markers from 9457 unigenes were generated and validated through genetic diversity analysis among 13 accessions of S. alterniflora. CONCLUSIONS: The present study explores the transcriptome of S. alterniflora to understand the gene regulation under salt stress in halophytes. The sequenced transcriptome (control and salt-regulated) of S. alterniflora provides a platform for further gene finding studies in grasses. This study and our previously published studies suggested that S. alterniflora is a rich reservoir of salt tolerance genes that can be used to develop salt tolerant cereal crops, especially rice, a major food crop of global importance.

Antiviral therapy with nucleotide/nucleoside analogues in chronic hepatitis B: A meta-analysis of prospective randomized trials.

Nucleotide/nucleoside analogues (antiviral therapy) are used in the therapy of HBeAg positive and HBeAg negative chronic hepatitis B. We analyzed ten selected randomized controlled with 2557 patients to estimate the effect of antiviral drugs in chronic hepatitis B with compared to placebo. Virological response, biochemical response, histological response, seroconversion of HBeAg, and loss of HBeAg were estimated as primary efficacy measures. The included studies were subjected for heterogeneity and publication bias. The heterogeneity was assessed with χ2 and I(2) statistics. Publication bias was assessed by funnel plot. Greater rates of improvement obtained in antiviral group for virological response [43.96 % vs. 3.15 %, RR = 0.57, 95 % CI = 0.54-0.61, p-value <0.00001], biochemical response [58.37 % vs. 21.87 %, RR = 0.52, 95 % CI = 0.48-0.56, p-value <0.00001], histological response [58.99 % vs. 27.13 %, RR = 0.56, 95 % CI = 0.50-0.63, p-value <0.0001], seroconversion of HBeAg [10.66 % vs. 5.56 %, RR = 0.94, 95 % CI = 0.91-0.97, p-value = 0.0005], and HBeAg loss [14.59 % vs. 9.64 %, RR = 0.92, 95 % CI = 0.88-0.96, p-value = 0.0002]. The safety analysis were carried out for adverse events such as headache [17.22 % vs. 17.34 %, OR = 1.09, 95 % CI = 0.81-1.46, p-value = 0.58], abdominal pain [16.46 % vs. 14.34 %, OR = 1.24, 95 % CI = 0.90-1.72, p-value = 0.19], and pharyngitis [22.22 % vs. 18.23 %, OR = 1.12, 95 % CI = 0.86-1.45, p-value = 0.40]. Excluding adverse events, all primary efficacy measures shown statistical significant result for chronic hepatitis treatment (p-value <0.05). Antiviral therapy provided significant benefit for the treatment of chronic hepatitis B with no measurable adverse effects.