18S rRNA gene amplicon sequencing combined with culture-based surveys of maize rhizosphere protists reveal dominant, plant-enriched and culturable community members.

Protists play important roles in shaping the microbial community of the rhizosphere and defining these roles will require the study of protist isolates. However, there is still a limited understanding of how well protist isolation efforts can capture the diversity and composition of rhizosphere protistan communities. Here, we report a simultaneous isolation and 18S rRNA gene amplicon sequencing survey describing the protist diversity of maize rhizospheres in two climatically and pedologically distinct sites. We demonstrated that the maize rhizosphere exerted significant and site-dependent effects on the protistan community structure and defined a set of core and rhizosphere-enriched protists. From the same root samples, we generated a library of 103 protist isolates representing 46 18S rRNA gene sequence variants from six eukaryotic supergroups. While cultured isolates represented a small proportion of total protist diversity recovered by sequencing, they included taxa enriched in rhizosphere soils across all samples, encompassing 9% of all core sequence variants. The isolation approach also captured 17 protists not detected through 18S rRNA gene amplicon sequencing. This study demonstrated that maize roots select for distinct protistan communities, and established a diverse protist culture collection that can be used for future research linking protists to rhizosphere status and plant health.

Foliar Application of Copper Oxide Nanoparticles Suppresses Fusarium Wilt Development on Chrysanthemum.

Micronutrients applied as nanoparticles of metal oxides have shown efficacy in vegetable and other crops for improving yield and reducing Fusarium diseases, but their role in ornamental crop management has not been investigated. In 2017, 2018, and 2020, nanoparticles of CuO, Mn2O3, or ZnO were foliarly applied at 500 µg/mL (0.6 mg/plant) to chrysanthemum transplants and planted in potting soil noninfested or infested with Fusarium oxysporum f. sp. chrysanthemi. An untreated control and a commercial fungicide, Fludioxonil, was also included. Chrysanthemums treated with nanoscale CuO had a 55, 30, and 32% reduction in disease severity ratings compared to untreated plants in 2017, 2018, and 2020, respectively. Specifically, the average dry biomass for the three years was reduced 22% by disease, but treatment with nanoscale CuO led to a 23% increase when compared to controls. Similar trends with plant height were observed. Horticultural quality was improved 28% with nano CuO and was equal to the fungicide. Nanoscale Mn2O3 and the fungicide did not consistently reduce disease ratings or increase dry biomass each year. Nanoscale ZnO was ineffective. Nanoscale CuO-treated plants had 24 to 48% more Cu/g tissue than controls (P < 0.001). These findings agree with past reports on food crops where single applications of nanoscale CuO improved plant health, growth, and yield and could offer significant impacts for managing plant diseases on ornamentals.

Pseudomonas syringae pv. phaseolicola Uses Distinct Modes of Stationary-Phase Persistence To Survive Bacteriocin and Streptomycin Treatments.

Antimicrobial treatment of bacteria often results in a small population of surviving tolerant cells, or persisters, that may contribute to recurrent infection. Antibiotic persisters are metabolically dormant, but the basis of their persistence in the presence of membrane-disrupting biological compounds is less well understood. We previously found that the model plant pathogen Pseudomonas syringae pv. phaseolicola 1448A (Pph) exhibits persistence to tailocin, a membrane-disrupting biocontrol compound with potential for sustainable disease control. Here, we compared physiological traits associated with persistence to tailocin and to the antibiotic streptomycin and established that both treatments leave similar frequencies of persisters. Microscopic profiling of treated populations revealed that while tailocin rapidly permeabilizes most cells, streptomycin treatment results in a heterogeneous population in the redox and membrane permeability state. Intact cells were sorted into three fractions according to metabolic activity, as indicated by a redox-sensing reporter dye. Streptomycin persisters were cultured from the fraction associated with the lowest metabolic activity, but tailocin persisters were cultured from a fraction associated with an active metabolic signal. Cells from culturable fractions were able to infect host plants, while the nonculturable fractions were not. Tailocin and streptomycin were effective in eliminating all persisters when applied sequentially, in addition to eliminating cells in other viable states. This study identifies distinct metabolic states associated with antibiotic persistence, tailocin persistence, and loss of virulence and demonstrates that tailocin is highly effective in eliminating dormant cells.IMPORTANCE Populations of genetically identical bacteria encompass heterogeneous physiological states. The small fraction of bacteria that are dormant can help the population survive exposure to antibiotics and other stresses, potentially contributing to recurring infection cycles in animal or plant hosts. Membrane-disrupting biological control treatments are effective in killing dormant bacteria, but these treatments also leave persister-like survivors. The current work demonstrates that in Pph, persisters surviving treatment with membrane-disrupting tailocin proteins have an elevated redox state compared to that of dormant streptomycin persisters. Combination treatment was effective in killing both persister types. Culturable persisters corresponded closely with infectious cells in each treated population, whereas the high-redox and unculturable fractions were not infectious. In linking redox states to heterogeneous phenotypes of tailocin persistence, streptomycin persistence, and infection capability, this work will inform the search for mechanisms and markers for each phenotype.

Induction of pre-chorismate, jasmonate and salicylate pathways by Burkholderia sp. RR18 in peanut seedlings.

AIMS: To characterize the mechanisms by which bacteria in the peanut rhizosphere promote plant growth and suppress Aspergillus niger, the fungus that causes collar rot of peanut. METHODS AND RESULTS: In all, 131 isolates cultured from the peanut rhizosphere were assayed for growth promotion in a seedling germination assay. The most effective isolate, RR18, was identified as Burkholderia sp. by 16S sequencing analysis. RR18 reduced collar rot disease incidence and increased the germination rate and biomass of peanut seeds, and had broad-spectrum antifungal activity. Quantitative analyses showed that RR18 induced long-lasting accumulation of jasmonic acid, salicylic acid and phenols, and triggered the activity of six defence enzymes related to these changes. Comparative proteomic analysis of treated and untreated seedlings revealed a clear induction of four abundant proteins, including a member of the pre-chorismate pathway, a regulator of clathrin-coated vesicles, a transcription factor and a hypothetical protein. CONCLUSION: Burkholderia sp. RR18 promotes peanut growth and disease resistance, and stably induces two distinct defence pathways associated with systemic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a strain of the Burkholderia cepacia complex can elicit both salicylic- and jasmonic-acid-mediated defences, in addition to having numerous other beneficial properties.

Activation of metabolic and stress responses during subtoxic expression of the type I toxin hok in Erwinia amylovora.

BACKGROUND: Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene and its cognate antitoxin. Several toxins are implied to affect the physiological state and stress tolerance of bacteria in a population. We previously identified a chromosomally encoded hok-sok type I TA system in Erwinia amylovora, the causative agent of fire blight disease on pome fruit trees. A high-level induction of the hok gene was lethal to E. amylovora cells through unknown mechanisms. The molecular targets or regulatory roles of Hok were unknown. RESULTS: Here, we examined the physiological and transcriptomic changes of Erwinia amylovora cells expressing hok at subtoxic levels that were confirmed to confer no cell death, and at toxic levels that resulted in killing of cells. In both conditions, hok caused membrane rupture and collapse of the proton motive force in a subpopulation of E. amylovora cells. We demonstrated that induction of hok resulted in upregulation of ATP biosynthesis genes, and caused leakage of ATP from cells only at toxic levels. We showed that overexpression of the phage shock protein gene pspA largely reversed the cell death phenotype caused by high levels of hok induction. We also showed that induction of hok at a subtoxic level rendered a greater proportion of stationary phase E. amylovora cells tolerant to the antibiotic streptomycin. CONCLUSIONS: We characterized the molecular mechanism of toxicity by high-level of hok induction and demonstrated that low-level expression of hok primes the stress responses of E. amylovora against further membrane and antibiotic stressors.

Genome Mining Shows Ubiquitous Presence and Extensive Diversity of Toxin-Antitoxin Systems in Pseudomonas syringae.

Bacterial toxin-antitoxin (TA) systems consist of two or more adjacent genes, encoding a toxin and an antitoxin. TA systems are implicated in evolutionary and physiological functions including genome maintenance, antibiotics persistence, phage defense, and virulence. Eight classes of TA systems have been described, based on the mechanism of toxin neutralization by the antitoxin. Although studied well in model species of clinical significance, little is known about the TA system abundance and diversity, and their potential roles in stress tolerance and virulence of plant pathogens. In this study, we screened the genomes of 339 strains representing the genetic and lifestyle diversity of the Pseudomonas syringae species complex for TA systems. Using bioinformatic search and prediction tools, including SLING, BLAST, HMMER, TADB2.0, and T1TAdb, we show that P. syringae strains encode 26 different families of TA systems targeting diverse cellular functions. TA systems in this species are almost exclusively type II. We predicted a median of 15 TA systems per genome, and we identified six type II TA families that are found in more than 80% of strains, while others are more sporadic. The majority of predicted TA genes are chromosomally encoded. Further functional characterization of the predicted TA systems could reveal how these widely prevalent gene modules potentially impact P. syringae ecology, virulence, and disease management practices.

Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing.

Long-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes constitute one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is NLR gene-rich. Here, toward identification of the Xo1 gene, we combined Nanopore and Illumina reads and generated a high-quality Carolina Gold Select genome assembly. We identified 529 complete or partial NLR genes and discovered, relative to Nipponbare, an expansion of NLR genes at the Xo1 locus. One of these has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain, and the repeats within each protein are nearly perfect. Across diverse Oryzeae, we identified two sub-clades of NLR genes with these features, varying in the presence of the zfBED domain and the number of repeats. The Carolina Gold Select genome assembly also uncovered at the Xo1 locus a rice blast resistance gene and a gene encoding a polyphenol oxidase (PPO). PPO activity has been used as a marker for blast resistance at the locus in some varieties; however, the Carolina Gold Select sequence revealed a loss-of-function mutation in the PPO gene that breaks this association. Our results demonstrate that whole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Finally, the Carolina Gold Select genome assembly will facilitate identification of other useful traits in this historically important variety.

Development of the Automated Primer Design Workflow Uniqprimer and Diagnostic Primers for the Broad-Host-Range Plant Pathogen Dickeya dianthicola.

Uniqprimer, a software pipeline developed in Python, was deployed as a user-friendly internet tool in Rice Galaxy for comparative genome analyses to design primer sets for PCRassays capable of detecting target bacterial taxa. The pipeline was trialed with Dickeya dianthicola, a destructive broad-host-range bacterial pathogen found in most potato-growing regions. Dickeya is a highly variable genus, and some primers available to detect this genus and species exhibit common diagnostic failures. Upon uploading a selection of target and nontarget genomes, six primer sets were rapidly identified with Uniqprimer, of which two were specific and sensitive when tested with D. dianthicola. The remaining four amplified a minority of the nontarget strains tested. The two promising candidate primer sets were trialed with DNA isolated from 116 field samples from across the United States that were previously submitted for testing. D. dianthicola was detected in 41 samples, demonstrating the applicability of our detection primers and suggesting widespread occurrence of D. dianthicola in North America.

Cell-length heterogeneity: a population-level solution to growth/virulence trade-offs in the plant pathogen Dickeya dadantii.

Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10µm), the rest elongated into filamentous cells (>10µm). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed upregulated metabolic genes and increased growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential production of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.

Chromosomally Encoded hok-sok Toxin-Antitoxin System in the Fire Blight Pathogen Erwinia amylovora: Identification and Functional Characterization.

Toxin-antitoxin (TA) systems are genetic elements composed of a protein toxin and a counteracting antitoxin that is either a noncoding RNA or protein. In type I TA systems, the antitoxin is a noncoding small RNA (sRNA) that base pairs with the cognate toxin mRNA interfering with its translation. Although type I TA systems have been extensively studied in Escherichia coli and a few human or animal bacterial pathogens, they have not been characterized in plant-pathogenic bacteria. In this study, we characterized a chromosomal locus in the plant pathogen Erwinia amylovora Ea1189 that is homologous to the hok-sok type I TA system previously identified in the Enterobacteriaceae-restricted plasmid R1. Phylogenetic analysis indicated that the chromosomal location of the hok-sok locus is, thus far, unique to E. amylovora We demonstrated that ectopic overexpression of hok is highly toxic to E. amylovora and that the sRNA sok reversed the toxicity of hok through mok, a reading frame presumably translationally coupled with hok We also identified the region that is essential for maintenance of the main toxicity of Hok. Through a hok-sok deletion mutant (Ea1189Δhok-sok), we determined the contribution of the hok-sok locus to cellular growth, micromorphology, and catalase activity. Combined, our findings indicate that the hok-sok TA system, besides being potentially self-toxic, provides fitness advantages to E. amylovoraIMPORTANCE Bacterial toxin-antitoxin systems have received great attention because of their potential as targets for antimicrobial development and as tools for biotechnology. Erwinia amylovora, the causal agent of fire blight disease on pome fruit trees, is a major plant-pathogenic bacterium. In this study, we identified and functionally characterized a unique chromosomally encoded hok-sok toxin-antitoxin system in E. amylovora that resembles the plasmid-encoded copies of this system in other Enterobacteriaceae This study of a type I toxin-antitoxin system in a plant-pathogenic bacterium provides the basis to further understand the involvement of toxin-antitoxin systems during infection by a plant-pathogenic bacterium. The new linkage between the hok-sok toxin-antitoxin system and the catalase-mediated oxidative stress response leads to additional considerations of targeting this system for antimicrobial development.

Survey of Toxin⁻Antitoxin Systems in Erwinia amylovora Reveals Insights into Diversity and Functional Specificity.

Toxin⁻antitoxin (TA) systems are diverse genetic modules with demonstrated roles in plasmid stability, stress management, biofilm formation and antibiotic persistence. However, relatively little is known about their functional significance in plant pathogens. In this study we characterize type II and IV TA systems in the economically important plant pathogen Erwinia amylovora. Hidden Markov Model (HMM) and BLAST-based programs were used to predict the identity and distribution of putative TA systems among sequenced genomes of E. amylovora and other plant-associated Erwinia spp. Of six conserved TA systems tested for function from E. amylovora, three (CbtA/CbeA, ParE/RHH and Doc/PhD) were validated as functional. CbtA was toxic to E. amylovora, but not to Escherichia coli. While the E. coli homolog of CbtA elicits the formation of lemon-shaped cells upon overexpression and targets cytoskeletal proteins FtsZ and MreB, E. amylovora CbtA led to cell elongation and did not interact with these cytoskeletal proteins. Phylogenetic analysis revealed that E. amylovora CbtA belongs to a distinct clade from the CbtA of pathogenic E. coli. This study expands the repertoire of experimentally validated TA systems in plant pathogenic bacteria, and suggests that the E. amylovora homolog of CbtA is functionally distinct from that of E. coli.

Regulation of Effector Delivery by Type III Secretion Chaperone Proteins in Erwinia amylovora.

Type III secretion (TTS) chaperones are critical for the delivery of many effector proteins from Gram-negative bacterial pathogens into host cells, functioning in the stabilization and hierarchical delivery of the effectors to the type III secretion system (TTSS). The plant pathogen Erwinia amylovora secretes at least four TTS effector proteins: DspE, Eop1, Eop3, and Eop4. DspE specifically interacts with the TTS chaperone protein DspF, which stabilizes the effector protein in the cytoplasm and promotes its efficient translocation through the TTSS. However, the role of E. amylovora chaperones in regulating the delivery of other secreted effectors is unknown. In this study, we identified functional interactions between the effector proteins DspE, Eop1, and Eop3 with the TTS chaperones DspF, Esc1 and Esc3 in yeast. Using site-directed mutagenesis, secretion, and translocation assays, we demonstrated that the three TTS chaperones have additive roles for the secretion and translocation of DspE into plant cells whereas DspF negatively affects the translocation of Eop1 and Eop3. Collectively, these results indicate that TTS chaperone proteins exhibit a cooperative behavior to orchestrate the effector secretion and translocation dynamics in E. amylovora.

The effector AvrRxo1 phosphorylates NAD in planta.

Gram-negative bacterial pathogens of plants and animals employ type III secreted effectors to suppress innate immunity. Most characterized effectors work through modification of host proteins or transcriptional regulators, although a few are known to modify small molecule targets. The Xanthomonas type III secreted avirulence factor AvrRxo1 is a structural homolog of the zeta toxin family of sugar-nucleotide kinases that suppresses bacterial growth. AvrRxo1 was recently reported to phosphorylate the central metabolite and signaling molecule NAD in vitro, suggesting that the effector might enhance bacterial virulence on plants through manipulation of primary metabolic pathways. In this study, we determine that AvrRxo1 phosphorylates NAD in planta, and that its kinase catalytic sites are necessary for its toxic and resistance-triggering phenotypes. A global metabolomics approach was used to independently identify 3'-NADP as the sole detectable product of AvrRxo1 expression in yeast and bacteria, and NAD kinase activity was confirmed in vitro. 3'-NADP accumulated upon transient expression of AvrRxo1 in Nicotiana benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae. Mutation of the catalytic aspartic acid residue D193 abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response. A mutation in the Walker A ATP-binding motif abolished the toxicity of AvrRxo1, but did not abolish the 3'-NADP production, virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that a type III effector targets the central metabolite and redox carrier NAD in planta, and that this catalytic activity is required for toxicity and suppression of the ROS burst.

Communication in the Phytobiome.

The phytobiome is composed of plants, their environment, and diverse interacting microscopic and macroscopic organisms, which together influence plant health and productivity. These organisms form complex networks that are established and regulated through nutrient cycling, competition, antagonism, and chemical communication mediated by a diverse array of signaling molecules. Integration of knowledge of signaling mechanisms with that of phytobiome members and their networks will lead to a new understanding of the fate and significance of these signals at the ecosystem level. Such an understanding could lead to new biological, chemical, and breeding strategies to improve crop health and productivity.

Characterization of the Xanthomonas translucens Complex Using Draft Genomes, Comparative Genomics, Phylogenetic Analysis, and Diagnostic LAMP Assays.

Prevalence of Xanthomonas translucens, which causes cereal leaf streak (CLS) in cereal crops and bacterial wilt in forage and turfgrass species, has increased in many regions in recent years. Because the pathogen is seedborne in economically important cereals, it is a concern for international and interstate germplasm exchange and, thus, reliable and robust protocols for its detection in seed are needed. However, historical confusion surrounding the taxonomy within the species has complicated the development of accurate and reliable diagnostic tools for X. translucens. Therefore, we sequenced genomes of 15 X. translucens strains representing six different pathovars and compared them with additional publicly available X. translucens genome sequences to obtain a genome-based phylogeny for robust classification of this species. Our results reveal three main clusters: one consisting of pv. cerealis, one consisting of pvs. undulosa and translucens, and a third consisting of pvs. arrhenatheri, graminis, phlei, and poae. Based on genomic differences, diagnostic loop-mediated isothermal amplification (LAMP) primers were developed that clearly distinguish strains that cause disease on cereals, such as pvs. undulosa, translucens, hordei, and secalis, from strains that cause disease on noncereal hosts, such as pvs. arrhenatheri, cerealis, graminis, phlei, and poae. Additional LAMP assays were developed that selectively amplify strains belonging to pvs. cerealis and poae, distinguishing them from other pathovars. These primers will be instrumental in diagnostics when implementing quarantine regulations to limit further geographic spread of X. translucens pathovars.

Suppression of Xo1-Mediated Disease Resistance in Rice by a Truncated, Non-DNA-Binding TAL Effector of Xanthomonas oryzae.

Delivered into plant cells by type III secretion from pathogenic Xanthomonas species, TAL (transcription activator-like) effectors are nuclear-localized, DNA-binding proteins that directly activate specific host genes. Targets include genes important for disease, genes that confer resistance, and genes inconsequential to the host-pathogen interaction. TAL effector specificity is encoded by polymorphic repeats of 33-35 amino acids that interact one-to-one with nucleotides in the recognition site. Activity depends also on N-terminal sequences important for DNA binding and C-terminal nuclear localization signals (NLS) and an acidic activation domain (AD). Coding sequences missing much of the N- and C-terminal regions due to conserved, in-frame deletions are present and annotated as pseudogenes in sequenced strains of Xanthomonas oryzae pv. oryzicola (Xoc) and pv. oryzae (Xoo), which cause bacterial leaf streak and bacterial blight of rice, respectively. Here we provide evidence that these sequences encode proteins we call "truncTALEs," for "truncated TAL effectors." We show that truncTALE Tal2h of Xoc strain BLS256, and by correlation truncTALEs in other strains, specifically suppress resistance mediated by the Xo1 locus recently described in the heirloom rice variety Carolina Gold. Xo1-mediated resistance is triggered by different TAL effectors from diverse X. oryzae strains, irrespective of their DNA binding specificity, and does not require the AD. This implies a direct protein-protein rather than protein-DNA interaction. Similarly, truncTALEs exhibit diverse predicted DNA recognition specificities. And, in vitro, Tal2h did not bind any of several potential recognition sites. Further, a single candidate NLS sequence in Tal2h was dispensable for resistance suppression. Many truncTALEs have one 28 aa repeat, a length not observed previously. Tested in an engineered TAL effector, this repeat required a single base pair deletion in the DNA, suggesting that it or a neighbor disengages. The presence of the 28 aa repeat, however, was not required for resistance suppression. TruncTALEs expand the paradigm for TAL effector-mediated effects on plants. We propose that Tal2h and other truncTALEs act as dominant negative ligands for an immune receptor encoded by the Xo1 locus, likely a nucleotide binding, leucine-rich repeat protein. Understanding truncTALE function and distribution will inform strategies for disease control.

AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental Contexts.

Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.

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.

Characterization of a novel clade of Xanthomonas isolated from rice leaves in Mali and proposal of Xanthomonas maliensis sp. nov.

Four bacterial strains, designated M89, M92, M97(T), and M106, were isolated in a previous study from surface-sterilized leaves of rice (Oryza sativa) or murainagrass (Ischaemum rugosum) at three sites in Mali, Africa. Here they were examined by a polyphasic taxonomic approach and analysis of a whole-genome sequence. Phylogenetic analyses based on 16S rRNA sequence and multilocus sequence analysis of seven genes showed that these four strains formed a distinct lineage representing a novel species within the genus Xanthomonas. This was supported by whole-genome average nucleotide identity values calculated from comparisons of strain M97(T) with established Xanthomonas species. The strains can be differentiated from the known Xanthomonas species on the basis of their fatty acid and carbohydrate utilization profiles. Population growth studies on rice confirmed that these bacteria multiply in rice leaves without causing symptoms. Identification of this novel species can be accomplished by using diagnostic primer sets or by gyrB gene sequence analysis. We propose to classify these rice- and grass-associated bacteria as Xanthomonas maliensis sp. nov. with strain M97(T) = CFBP7942(T) = LMG27592(T) as the type strain.

Sensitive detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by loop-mediated isothermal amplification.

Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10(4) to 10(5) CFU ml(-1), while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.