Three de novo assembled wild cacao genomes from the Upper Amazon.

Theobroma cacao, the chocolate tree, is indigenous to the Amazon basin, the greatest biodiversity hotspot on earth. Recent advancement in plant genomics highlights the importance of de novo sequencing of multiple reference genomes to capture the genome diversity present in different cacao populations. In this study, three high-quality chromosome-level genomes of wild cacao were constructed, de novo assembled with HiFi long reads sequencing, and scaffolded using a reference-free strategy. These genomes represent the three most important genetic clusters of cacao trees from the Upper Amazon region. The three wild cacao genomes were compared with two reference genomes of domesticated cacao. The five cacao genetic clusters were inferred to have diverged in the early and middle Pleistocene period, approximately 1.83-0.69 million years ago. The results shown here serve as an example of understanding how the Amazonian biodiversity was developed. The three wild cacao genomes provide valuable resources for studying genetic diversity and advancing genetic improvement of this species.

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Utilization of a New Hundred-Genomes Pipeline to Design a Rapid Duplex LAMP Detection Assay for Xanthomonas euvesicatoria and X. vesicatoria in Tomato.

Xanthomonas euvesicatoria and X. vesicatoria are two economically important causal agents of bacterial spot (BS) of tomato and pepper. Management of BS in the field requires rapid and accurate detection. Therefore, this work aimed to develop a pipeline to design a simple, fast, and reliable assay for the detection of X. euvesicatoria and X. vesicatoria by loop-mediated isothermal amplification. In total, 109 publicly available whole genomic sequences of 24 different species of bacterial pathogens were used to design primers that would amplify the DNA of the two target species. Laboratory testing of the assay was performed on pure bacterial cultures and artificially infected plants, and amplification was conducted with both a sophisticated laboratory instrument and a simple mobile platform. The testing of the assay confirmed its specificity with a sensitivity reaching 1 pg µl-1 for both pathogens with an assay duration of 40 min on a mobile detection platform. Our diagnostics development pipeline enables the easy and fast design of a reliable detection assay in the genomics age. By validating the pipeline with X. euvesicatoria and X. vesicatoria pathogens, we have simultaneously developed an assay with high specificity, sensitivity, and speed, which will allow it to be deployed, contributing to successful management of BS.

United States tea: A synopsis of ongoing tea research and solutions to United States tea production issues.

Tea is a steeped beverage made from the leaves of Camellia sinensis. Globally, this healthy, caffeine-containing drink is one of the most widely consumed beverages. At least 50 countries produce tea and most of the production information and tea research is derived from international sources. Here, we discuss information related to tea production, genetics, and chemistry as well as production issues that affect or are likely to affect emerging tea production and research in the United States. With this review, we relay current knowledge on tea production, threats to tea production, and solutions to production problems to inform this emerging market in the United States.

KEC: unique sequence search by K-mer exclusion.

SUMMARY: Searching for amino acid or nucleic acid sequences unique to one organism may be challenging depending on size of the available datasets. K-mer elimination by cross-reference (KEC) allows users to quickly and easily find unique sequences by providing target and non-target sequences. Due to its speed, it can be used for datasets of genomic size and can be run on desktop or laptop computers with modest specifications. AVAILABILITY AND IMPLEMENTATION: KEC is freely available for non-commercial purposes. Source code and executable binary files compiled for Linux, Mac and Windows can be downloaded from https://github.com/berybox/KEC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Rubisco activase A (RcaA) is a central node in overlapping gene network of drought and salinity in Barley (Hordeum vulgare L.) and may contribute to combined stress tolerance.

Co-occurrence of abiotic stresses, especially drought and salinity, is a natural phenomenon in field conditions and is worse for crop production than any single stress. Nowadays, rigorous methods of meta-analysis and systems biology have made it possible to perform cross-study comparisons of single stress experiments, which can uncover main overlapping mechanisms underlying tolerance to combined stress. In this study, a meta-analysis of RNA-Seq data was conducted to obtain the overlapping gene network of drought and salinity stresses in barley (Hordeum vulgare L.), which identified Rubisco activase A (RcaA) as a hub gene in the dual-stress response. Thereafter, a greenhouse experiment was carried out using two barley genotypes with different abiotic stress tolerance and evaluated several physiochemical properties as well as the expression profile and protein activity of RcaA. Finally, machine learning analysis was applied to uncover relationships among combined stress tolerance and evaluated properties. We identified 441 genes which were differentially expressed under both drought and salinity stress. Results revealed that the photosynthesis pathway and, in particular, the RcaA gene are major components of the dual-stress responsive transcriptome. Comparative physiochemical and molecular evaluations further confirmed that enhanced photosynthesis capability, mainly through regulation of RcaA expression and activity as well as accumulation of proline content, have a significant association with combined drought and salinity stress tolerance in barley. Overall, our results clarify the importance of RcaA in combined stress tolerance and may provide new insights for future investigations.

The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads.

Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.

Development of Real-Time and Colorimetric Loop Mediated Isothermal Amplification Assay for Detection of Xanthomonas gardneri.

Xanthomonas gardneri is one of the causal agents of bacterial spot (BS), an economically important bacterial disease of tomato and pepper. Field-deployable and portable loop-mediated isothermal amplification (LAMP)-based instruments provide rapid and sensitive detection of plant pathogens. In order to rapidly and accurately identify and differentiate X. gardneri from other BS-causing Xanthomonas spp., we optimized a new real-time monitoring LAMP-based method targeting the X. gardneri-specific hrpB gene. Specificity and sensitivity of real-time and colorimetric LAMP assays were tested on the complex of bacterial strains pathogenic to tomato and pepper and on plants infected by the pathogen. The assay detection limit was 1 pg/µL of genomic DNA with an assay duration of only 30 min. The use of portable and handheld instruments allows for fast analysis, reducing the diagnosis time, and can contribute to proper disease management and control of X. gardneri. Due to the high efficiency of this method, we suggest its use as a standard diagnostic tool during phytosanitary controls.

High temperature-induced plant disease susceptibility: more than the sum of its parts.

Higher temperatures associated with climate change often increase the severity of plant diseases. An understanding of how plants respond to pathogens during high temperature stress is required for crop improvement, but the molecular mechanisms underlying this response are largely unknown. Mechanistic research has primarily focused on plant responses during either single stresses or heat-induced loss of single gene resistance. Transcriptome analyses of plant responses to a single stress compared to combined-stresses reveal significant differences showing that single-stress response studies are inadequate for determining the mechanisms of high temperature-induced disease susceptibility. To combat plant disease in light of climate change, future research will require comprehensive study designs and analyses.

High-Quality Genome Resource of Xanthomonas hyacinthi Generated via Long-Read Sequencing.

The bacterial plant pathogen Xanthomonas hyacinthi is the causal agent of yellow disease of Hyacinthus and other ornamental plant genera. There is no available complete genome for X. hyacinthi, limiting basic research for this pathogen. Here, we release a high-quality complete genome sequence for the X. hyacinthi type strain, CFBP 1156. Single-molecule real-time (SMRT) sequencing with a mean coverage of 306× revealed two contigs of 4,918,645 and 44,381 bp in size. This was the first characterized plant-disease-causing species of Xanthomonas and this genome provides a resource to better understand the biology of yellow disease of hyacinth.

Abiotic and biotic stresses induce a core transcriptome response in rice.

Environmental stresses greatly limit crop yield. With the increase in extreme weather events due to climate change and the constant pressure of diseases and pests, there is an urgent need to develop crop varieties that can tolerate multiple stresses. However, our knowledge of how plants broadly respond to stress is limited. Here, we explore the rice core stress response via meta-analysis of publicly available rice transcriptome data. Our results confirm that rice universally down-regulates photosynthesis in response to both abiotic and biotic stress. Rice also generally up-regulates hormone-responsive genes during stress response, most notably genes in the abscisic acid, jasmonic acid and salicylic acid pathways. We identified several promoter motifs that are likely involved in stress-responsive regulatory mechanisms in rice. With this work, we provide a list of candidate genes to study for improving rice stress tolerance in light of environmental stresses. This work also serves as a proof of concept to show that meta-analysis of diverse transcriptome data is a valid approach to develop robust hypotheses for how plants respond to stress.

In Planta Bacterial Transcriptomics Predict Plant Disease Outcomes.

Plants are colonized by pathogenic and beneficial microbes. When pathogenic bacteria contact plant cells, bacterial gene expression changes, influencing disease development. The impact of host plant immunity on bacterial gene expression remains largely unexplored. Recent studies show how in planta bacterial transcriptomics methods can better define mechanisms of plant-microbe interactions.

RNA-Seq analysis reveals insight into enhanced rice Xa7-mediated bacterial blight resistance at high temperature.

Plant disease is a major challenge to agriculture worldwide, and it is exacerbated by abiotic environmental factors. During some plant-pathogen interactions, heat stress allows pathogens to overcome host resistance, a phenomenon which could severely impact crop productivity considering the global warming trends associated with climate change. Despite the importance of this phenomenon, little is known about the underlying molecular mechanisms. To better understand host plant responses during simultaneous heat and pathogen stress, we conducted a transcriptomics experiment for rice plants (cultivar IRBB61) containing Xa7, a bacterial blight disease resistance (R) gene, that were infected with Xanthomonas oryzae, the bacterial blight pathogen of rice, during high temperature stress. Xa7-mediated resistance is unusual relative to resistance mediated by other R genes in that it functions better at high temperatures. Using RNA-Seq technology, we identified 8,499 differentially expressed genes as temperature responsive in rice cultivar IRBB61 experiencing susceptible and resistant interactions across three time points. Notably, genes in the plant hormone abscisic acid biosynthesis and response pathways were up-regulated by high temperature in both mock-treated plants and plants experiencing a susceptible interaction and were suppressed by high temperature in plants exhibiting Xa7-mediated resistance. Genes responsive to salicylic acid, an important plant hormone for disease resistance, were down-regulated by high temperature during both the susceptible and resistant interactions, suggesting that enhanced Xa7-mediated resistance at high temperature is not dependent on salicylic acid signaling. A DNA sequence motif similar to known abscisic acid-responsive cis-regulatory elements was identified in the promoter region upstream of genes up-regulated in susceptible but down-regulated in resistant interactions. The results of our study suggest that the plant hormone abscisic acid is an important node for cross-talk between plant transcriptional response pathways to high temperature stress and pathogen attack. Genes in this pathway represent an important focus for future study to determine how plants evolved to deal with simultaneous abiotic and biotic stresses.

A resistance locus in the American heirloom rice variety Carolina Gold Select is triggered by TAL effectors with diverse predicted targets and is effective against African strains of Xanthomonas oryzae pv. oryzicola.

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X. oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes.