Xanthomonas as a model system for studying pathogen emergence and evolution.

In this review, we highlight studies where whole genome sequencing, comparative genomics and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understanding of adaptive evolution of secretion systems and their effectors. We focus on Xanthomonas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species, and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context.

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani.

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.

Genetic and transcriptomic dissection of host defense to Goss's bacterial wilt and leaf blight of maize.

Goss's wilt, caused by the Gram-positive actinobacterium Clavibacter nebraskensis, is an important bacterial disease of maize. The molecular and genetic mechanisms of resistance to the bacterium, or, in general, Gram-positive bacteria causing plant diseases, remain poorly understood. Here, we examined the genetic basis of Goss's wilt through differential gene expression, standard genome-wide association mapping (GWAS), extreme phenotype (XP) GWAS using highly resistant (R) and highly susceptible (S) lines, and quantitative trait locus (QTL) mapping using 3 bi-parental populations, identifying 11 disease association loci. Three loci were validated using near-isogenic lines or recombinant inbred lines. Our analysis indicates that Goss's wilt resistance is highly complex and major resistance genes are not commonly present. RNA sequencing of samples separately pooled from R and S lines with or without bacterial inoculation was performed, enabling identification of common and differential gene responses in R and S lines. Based on expression, in both R and S lines, the photosynthesis pathway was silenced upon infection, while stress-responsive pathways and phytohormone pathways, namely, abscisic acid, auxin, ethylene, jasmonate, and gibberellin, were markedly activated. In addition, 65 genes showed differential responses (up- or down-regulated) to infection in R and S lines. Combining genetic mapping and transcriptional data, individual candidate genes conferring Goss's wilt resistance were identified. Collectively, aspects of the genetic architecture of Goss's wilt resistance were revealed, providing foundational data for mechanistic studies.

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Immune phenotype is differentially affected by changing the type of bovine respiratory disease vaccine administered at revaccination in beef heifers.

During preconditioning, modified-live vaccines are frequently administered to beef calves before weaning. In this study, we began to characterize the immune phenotype of calves that received a modified-live vaccination at 3-4 months of age and then either received the same modified-live or an inactivated vaccine upon arrival at the feedlot (weaning) and 28 days post-arrival (booster). Innate and adaptive immune measures were assessed before revaccination and 14 and 28 days post. Heifers that received three doses of the modified-live vaccine exhibited a relatively balanced immune response based on increases in mean cytokine concentrations (IL-17, IL-21) and total immunoglobulin-G (IgG) and subsets IgG1 and IgG2, which are related to both arms of the adaptive immune system. Conversely, heifers that received one dose of modified live and two doses of the inactivated vaccine had a more robust neutrophil chemotactic response and greater serum-neutralizing antibody titers, resulting in an enhanced innate immune and a skewed proinflammatory response. These results indicate that the revaccination protocol used after initial vaccination with a modified-live vaccine differentially influences the immune phenotype of beef calves, with three doses of modified live inducing potentially immune homeostasis and a combination of modified live and inactivated vaccines inducing a skewed immune phenotype. However, more research is needed to determine the protective efficacy of these vaccination protocols against disease.

Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria.

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

The Dynamic Transcription Activator-Like Effector Family of Xanthomonas.

Transcription activator-like effectors (TALEs) are bacterial proteins that are injected into the eukaryotic nucleus to act as transcriptional factors and function as key virulence factors of the phytopathogen Xanthomonas. TALEs are translocated into plant host cells via the type III secretion system and induce the expression of host susceptibility (S) genes to facilitate disease. The unique modular DNA binding domains of TALEs comprise an array of nearly identical direct repeats that enable binding to DNA targets based on the recognition of a single nucleotide target per repeat. The very nature of TALE structure and function permits the proliferation of TALE genes and evolutionary adaptations in the host to counter TALE function, making the TALE-host interaction the most dynamic story in effector biology. The TALE genes appear to be a relatively young effector gene family, with a presence in all virulent members of some species and absent in others. Genome sequencing has revealed many TALE genes throughout the xanthomonads, and relatively few have been associated with a cognate S gene. Several species, including Xanthomonas oryzae pv. oryzae and X. citri pv. citri, have near absolute requirement for TALE gene function, while the genes appear to be just now entering the disease interactions with new fitness contributions to the pathogens of tomato and pepper among others. Deciphering the simple and effective DNA binding mechanism also has led to the development of DNA manipulation tools in fields of gene editing and transgenic research. In the three decades since their discovery, TALE research remains at the forefront of the study of bacterial evolution, plant-pathogen interactions, and synthetic biology. We also discuss critical questions that remain to be addressed regarding TALEs.

Meta-analysis of the effects of monensin on performance of beef replacement heifers and beef cows.

Although performance benefits of monensin have been extensively studied in finishing cattle, growing cattle, and dairy cows, considerably less published work is available evaluating response to monensin supplementation in cow-calf production systems. This meta-analysis investigated the impacts of monensin on performance of beef cows and developing replacement heifers. The replacement heifer analysis was conducted using data from 18 different peer-reviewed publications and experiment station reports. The mature cow analysis included 21 different publications and experiment station reports. The metaphor package (version 2.4-0; Viechtbauer, 2010) for R (version 4.0.3; www.r-project.org) was used to determine the overall effect size of monensin compared to a negative control. Each study's n, means, and SEM or P value was used to calculate the mean difference and estimate of within study variance for responses of interest. In replacement heifers, monensin treatment increased (P < 0.01); average daily gain (+0.03 ± 0.008 kg/d), feed efficiency (+0.013 ± 0.008 gain:feed), and percentage cycling before the breeding season (+15.9 ± 5.13%); while decreasing (P < 0.01): dry matter intake (0.293 ± 0.081 kg), and age at puberty (-8.9 ± 1.48 d). Six studies reporting ad libitum forage intake for mature cows showed decreased (P = 0.008) DMI by 0.85 ± 0.32 kg/d. Six studies reported milk yield and revealed an increase (P = 0.01) of 0.39 ± 0.15 kg/d when cows were supplemented with monensin. Monensin supplementation resulted in a reduction (P = 0.02) in days to first estrus by 18 ± 8.2 d and percentage of cows exhibiting estrus prior to the breeding season was increased by 19 ± 8% (P = 0.03). There were no differences in artificial insemination pregnancy nor total pregnancy for either the heifer or mature cow data sets. This analysis indicates potential for use of monensin in heifer development and beef cow production systems. Further research is needed to elucidate the effects on reproductive efficiency, DMI, milk production, weight, and body composition change.

Redox-engineering enhances maize thermotolerance and grain yield in the field.

Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a sixfold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment.

Meta-analysis of the effects of monensin on growth and bloat of cattle on pasture.

Monensin has been part of the beef production landscape for over 45 years. Although first approved for use in finishing cattle, it has since been approved for cattle in extensive production systems and has been an economical way to increase performance of forage-fed animals. This meta-analysis investigated the impacts of monensin on performance of stocker cattle on high-forage diets. The stocker performance analysis resulted from 38 experiments with 73 mean comparisons; bloat analysis was conducted with 12 experiments with 23 mean comparisons. The metaphor package (version 2.4-0) for R (version 4.0.3; www.r-project.org) was used to determine the overall effect size of monensin compared to a negative control. Each study's n, means, and SEM or P-value was used to calculate the mean difference and estimate of within-study variance for responses of interest. Moderators of monensin response considered in the analysis were delivery method, dose, study duration, initial calf BW, diet ME and CP, and forage category. Initial BW and basal ADG averaged 236 ± 45.9 kg and 0.72 ± 0.28 kg, respectively. In the ADG analysis, the only significant moderator of those considered was length of the study (P < 0.01); as duration of the study increased, the ADG response to monensin decreased by 0.0007 kg/day. For the average 112-day length of study, the average monensin response was estimated to be 0.0784 kg/day increase in ADG, approximately 10% above controls. Sufficient information was presented in 18 citations representing 40 mean comparisons for determining the effect of monensin on BW at the end of the experiment. The response model (P < 0.01) for ending BW, kg = 22.3-0.05 (initial calf BW, kg). Thus, for the average initial BW of 235 kg the average monensin response was estimated to be 10.6 kg increase in average ending BW. The incidence (-20%) and severity (-0.7 bloat score) of bloat was found to be reduced in bloat-prone pastures. There is ample evidence that monensin increases performance of growing calves on high forage diets along with reducing the incidence and severity of bloat.

Chromosome-level genome assembly of a regenerable maize inbred line A188.

BACKGROUND: The maize inbred line A188 is an attractive model for elucidation of gene function and improvement due to its high embryogenic capacity and many contrasting traits to the first maize reference genome, B73, and other elite lines. The lack of a genome assembly of A188 limits its use as a model for functional studies. RESULTS: Here, we present a chromosome-level genome assembly of A188 using long reads and optical maps. Comparison of A188 with B73 using both whole-genome alignments and read depths from sequencing reads identify approximately 1.1 Gb of syntenic sequences as well as extensive structural variation, including a 1.8-Mb duplication containing the Gametophyte factor1 locus for unilateral cross-incompatibility, and six inversions of 0.7 Mb or greater. Increased copy number of carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression in seeds together with low expression of yellow endosperm 1 (y1) reduces carotenoid accumulation, accounting for the white seed phenotype of A188. Furthermore, transcriptome and epigenome analyses reveal enhanced expression of defense pathways and altered DNA methylation patterns of the embryonic callus. CONCLUSIONS: The A188 genome assembly provides a high-resolution sequence for a complex genome species and a foundational resource for analyses of genome variation and gene function in maize. The genome, in comparison to B73, contains extensive intra-species structural variations and other genetic differences. Expression and network analyses identify discrete profiles for embryonic callus and other tissues.

Ectopic Expression of a Heterologous Glutaredoxin Enhances Drought Tolerance and Grain Yield in Field Grown Maize.

Drought stress is a major constraint in global maize production, causing almost 30-90% of the yield loss depending upon growth stage and the degree and duration of the stress. Here, we report that ectopic expression of Arabidopsis glutaredoxin S17 (AtGRXS17) in field grown maize conferred tolerance to drought stress during the reproductive stage, which is the most drought sensitive stage for seed set and, consequently, grain yield. AtGRXS17-expressing maize lines displayed higher seed set in the field, resulting in 2-fold and 1.5-fold increase in yield in comparison to the non-transgenic plants when challenged with drought stress at the tasseling and silking/pollination stages, respectively. AtGRXS17-expressing lines showed higher relative water content, higher chlorophyll content, and less hydrogen peroxide accumulation than wild-type (WT) control plants under drought conditions. AtGRXS17-expressing lines also exhibited at least 2-fold more pollen germination than WT plants under drought stress. Compared to the transgenic maize, WT controls accumulated higher amount of proline, indicating that WT plants were more stressed over the same period. The results present a robust and simple strategy for meeting rising yield demands in maize under water limiting conditions.

The Xa7 resistance gene guards the rice susceptibility gene SWEET14 against exploitation by the bacterial blight pathogen.

Many plant disease resistance (R) genes function specifically in reaction to the presence of cognate effectors from a pathogen. Xanthomonas oryzae pathovar oryzae (Xoo) uses transcription activator-like effectors (TALes) to target specific rice genes for expression, thereby promoting host susceptibility to bacterial blight. Here, we report the molecular characterization of Xa7, the cognate R gene to the TALes AvrXa7 and PthXo3, which target the rice major susceptibility gene SWEET14. Xa7 was mapped to a unique 74-kb region. Gene expression analysis of the region revealed a candidate gene that contained a putative AvrXa7 effector binding element (EBE) in its promoter and encoded a 113-amino-acid peptide of unknown function. Genome editing at the Xa7 locus rendered the plants susceptible to avrXa7-carrying Xoo strains. Both AvrXa7 and PthXo3 activated a GUS reporter gene fused with the EBE-containing Xa7 promoter in Nicotiana benthamiana. The EBE of Xa7 is a close mimic of the EBE of SWEET14 for TALe-induced disease susceptibility. Ectopic expression of Xa7 triggers cell death in N. benthamiana. Xa7 is prevalent in indica rice accessions from 3000 rice genomes. Xa7 appears to be an adaptation that protects against pathogen exploitation of SWEET14 and disease susceptibility.

Big Genes, Small Effectors: Pea Aphid Cassette Effector Families Composed From Miniature Exons.

Aphids secrete proteins from their stylets that evidence indicates function similar to pathogen effectors for virulence. Here, we describe two small candidate effector gene families of the pea aphid, Acyrthosiphon pisum, that share highly conserved secretory signal peptide coding regions and divergent non-secretory coding sequences derived from miniature exons. The KQY candidate effector family contains eleven members with additional isoforms, generated by alternative splicing. Pairwise comparisons indicate possible four unique KQY families based on coding regions without the secretory signal region. KQY1a, a representative of the family, is encoded by a 968 bp mRNA and a gene that spans 45.7 kbp of the genome. The locus consists of 37 exons, 33 of which are 15 bp or smaller. Additional KQY members, as well as members of the KHI family, share similar features. Differential expression analyses indicate that the genes are expressed preferentially in salivary glands. Proteomic analysis on salivary glands and saliva revealed 11 KQY members in salivary proteins, and KQY1a was detected in an artificial diet solution after aphid feeding. A single KQY locus and two KHI loci were identified in Myzus persicae, the peach aphid. Of the genes that can be anchored to chromosomes, loci are mostly scattered throughout the genome, except a two-gene region (KQY4/KQY6). We propose that the KQY family expanded in A. pisum through combinatorial assemblies of a common secretory signal cassette and novel coding regions, followed by classical gene duplication and divergence.

Xanthomonas diversity, virulence and plant-pathogen interactions.

Xanthomonas spp. encompass a wide range of plant pathogens that use numerous virulence factors for pathogenicity and fitness in plant hosts. In this Review, we examine recent insights into host-pathogen co-evolution, diversity in Xanthomonas populations and host specificity of Xanthomonas spp. that have substantially improved our fundamental understanding of pathogen biology. We emphasize the virulence factors in xanthomonads, such as type III secreted effectors including transcription activator-like effectors, type II secretion systems, diversity resulting in host specificity, evolution of emerging strains, activation of susceptibility genes and strategies of host evasion. We summarize the genomic diversity in several Xanthomonas spp. and implications for disease outbreaks, management strategies and breeding for disease resistance.

Early Drought-Responsive Genes Are Variable and Relevant to Drought Tolerance.

Drought stress is an important crop yield limiting factor worldwide. Plant physiological responses to drought stress are driven by changes in gene expression. While drought-responsive genes (DRGs) have been identified in maize, regulation patterns of gene expression during progressive water deficits remain to be elucidated. In this study, we generated time-series transcriptomic data from the maize inbred line B73 under well-watered and drought conditions. Comparisons between the two conditions identified 8,626 DRGs and the stages (early, middle, and late drought) at which DRGs occurred. Different functional groups of genes were regulated at the three stages. Specifically, early and middle DRGs display higher copy number variation among diverse Zea mays lines, and they exhibited stronger associations with drought tolerance as compared to late DRGs. In addition, correlation of expression between small RNAs (sRNAs) and DRGs from the same samples identified 201 negatively sRNA/DRG correlated pairs, including genes showing high levels of association with drought tolerance, such as two glutamine synthetase genes, gln2 and gln6 The characterization of dynamic gene responses to progressive drought stresses indicates important adaptive roles of early and middle DRGs, as well as roles played by sRNAs in gene expression regulation upon drought stress.

Genomic Acquisitions in Emerging Populations of Xanthomonas vasicola pv. vasculorum Infecting Corn in the United States and Argentina.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

Factorial estimating assembly base errors using k-mer abundance difference (KAD) between short reads and genome assembled sequences.

Genome sequences provide genomic maps with a single-base resolution for exploring genetic contents. Sequencing technologies, particularly long reads, have revolutionized genome assemblies for producing highly continuous genome sequences. However, current long-read sequencing technologies generate inaccurate reads that contain many errors. Some errors are retained in assembled sequences, which are typically not completely corrected by using either long reads or more accurate short reads. The issue commonly exists, but few tools are dedicated for computing error rates or determining error locations. In this study, we developed a novel approach, referred to as k-mer abundance difference (KAD), to compare the inferred copy number of each k-mer indicated by short reads and the observed copy number in the assembly. Simple KAD metrics enable to classify k-mers into categories that reflect the quality of the assembly. Specifically, the KAD method can be used to identify base errors and estimate the overall error rate. In addition, sequence insertion and deletion as well as sequence redundancy can also be detected. Collectively, KAD is valuable for quality evaluation of genome assemblies and, potentially, provides a diagnostic tool to aid in precise error correction. KAD software has been developed to facilitate public uses.

Recent advances in developing disease resistance in plants.

Approaches to manipulating disease resistance in plants is expanding exponentially due to advances in our understanding of plant defense mechanisms and new tools for manipulating the plant genome. The application of effective strategies is only limited now by adoption of rapid classical genetic techniques and the acceptance of genetically engineered traits for some problems. The use of genome editing and cis-genetics, where possible, may facilitate applications that otherwise require considerable time or genetic engineering, depending on settling legal definitions of the products. Nonetheless, the variety of approaches to developing disease resistance has never been greater.

A New TALe to Tell?

In this issue of Cell Host & Microbe, Wu et al. (2019) propose that a bacterial type III effector modifies the host milieu specifically to inhibit competing pathogens with little effect on the inciting pathogen populations. The work raises awareness to examine effectors for niche modification and has implications for the microbial community structure.