Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity.

Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

Expression of the Type III Secretion System Genes in Epiphytic Erwinia amylovora Cells on Apple Stigmas Benefits Endophytic Infection at the Hypanthium.

Erwinia amylovora causes fire blight on rosaceous plants. One of the major entry points of E. amylovora into hosts is flowers, where E. amylovora proliferates epiphytically on stigmatic and hypanthium surfaces and, subsequently, causes endophytic infection at the hypanthium. The type III secretion system (T3SS) is an important virulence factor in E. amylovora. Although the role of T3SS during endophytic infection is well characterized, its expression during epiphytic colonization and role in the subsequent infection is less understood. Here, we investigated T3SS gene expression in epiphytic E. amylovora on stigma and hypanthium of apple flowers under different relative humidities (RH). On stigma surfaces, T3SS was expressed in a high percentage of E. amylovora cells, and its expression promoted epiphytic growth. On hypanthium surfaces, however, T3SS was expressed in fewer E. amylovora cells than on the stigma, and displayed no correlation with epiphytic growth, even though T3SS expression is essential for infection. E. amylovora cells grown on stigmatic surfaces and then flushed down to the hypanthium displayed a higher level of T3SS expression than cells grown on the hypanthium surface alone. Furthermore, E. amylovora cells precultured on stigma had a higher potential to infect flowers than E. amylovora cells precultured in a T3SS-repressive medium. This suggests that T3SS induction during the stigmatic epiphytic colonization may be beneficial for subsequent infection. Finally, epiphytic expression of T3SS was influenced by RH. Higher percentage of stigmatic E. amylovora cells expressed T3SS under high RH than under low RH.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Involvement of non-coding RNAs during infection of rice by Acidovorax oryzae.

The expression of non-coding RNAs (ncRNAs) has been observed in a variety of bacteria. However, the function of ncRNAs and their regulatory targets are largely unknown, and few ncRNAs are found to be associated with bacterial virulence. The bacterial brown stripe pathogen Acidovorax oryzae (Ao) RS-1 shows a high level of condition-dependent differential expression of ncRNA, which we identified in a genome wide screen. We experimentally validated 66 differentially expressed ncRNAs using an integrative analysis of conservative genome sequences and transcriptomic data during in vivo interaction of the bacterial pathogen with the rice plant. To test the relevance of the differentially expressed ncRNAs, we chose four with different positions within the genome, and with different secondary structures and promoter activities. The results show that the overexpression of the four ncRNAs caused a significant change in virulence-related phenotypes, resistance to various environmental stresses, expression of secretion systems and effector proteins, while changing the expression of ncRNA putative target genes. We conclude that these ncRNAs are examples for the inherent regulatory roles for many of the observed ncRNAs in response to changing conditions such as host interaction or environmental adaption.

Complete Genome Sequences of Curtobacterium, Pantoea, Erwinia, and Two Pseudomonas sp. Strains, Isolated from Apple Flower Stigmas from Connecticut, USA.

The genome sequences of 5 bacterial strains isolated from apple flower stigmas are reported. The strains represent species of Curtobacterium, Pantoea, and Erwinia and two species of Pseudomonas These data will provide information for future taxonomic studies and information for investigating the metabolic and functional characteristics of apple flower-colonizing bacteria.

Genome Sequence and Adaptation Analysis of the Human and Rice Pathogenic Strain Burkholderia glumae AU6208.

Burkholderia glumae causes rice (Oryza sativa) bacterial panicle blight, which is an increasingly economically important disease worldwide. As the first B. glumae strain isolated from patients with chronic infections, AU6208 has been reported as an opportunistic clinic pathogen. However, our understanding of the molecular mechanism underlying human pathogenesis by B. glumae remains rudimentary. In this study, we report the complete genome sequence of the human-isolated B. glumae strain AU6208 and compare this to the genome of the rice-pathogenic B. glumae type strain LMG 2196T. Analysis of the average nucleotide identity demonstrated 99.4% similarity between the human- and plant-pathogenic strains. However, the phenotypic results from this study suggest a history of niche adaptation and divergence. In particular, we found 44 genes were predicted to be horizontally transferred into AU6208, and most of these genes were upregulated in conditions that mimic clinical conditions. In these, the gene pair sbnAB encodes key enzymes in antibiotic biosynthesis. These results suggest that horizontal gene transfer in AU6208 may be responsible for selective advantages in its pathogenicity in humans. Our analysis of the AU6208 genome and comparison with that of LMG 2196T reveal the evolutionary signatures of B. glumae in the process of switching niches from plants to humans.

Temporal and spatial dynamics in the apple flower microbiome in the presence of the phytopathogen Erwinia amylovora.

Plant microbiomes have important roles in plant health and productivity. However, despite flowers being directly linked to reproductive outcomes, little is known about the microbiomes of flowers and their potential interaction with pathogen infection. Here, we investigated the temporal spatial dynamics of the apple stigma microbiome when challenged with a phytopathogen Erwinia amylovora, the causal agent of fire blight disease. We profiled the microbiome from the stigmas of individual flowers, greatly increasing the resolution at which we can characterize shifts in the composition of the microbiome. Individual flowers harbored unique microbiomes at the operational taxonomic unit level. However, taxonomic analysis of community succession showed a population gradually dominated by bacteria within the families Enterobacteriaceae and Pseudomonadaceae. Flowers inoculated with E. amylovora established large populations of the phytopathogen, with pathogen-specific gene counts of >3.0 × 107 in 90% of the flowers. Yet, only 42% of inoculated flowers later developed fire blight symptoms. This reveals that pathogen abundance on the stigma is not sufficient to predict disease outcome. Our data demonstrate that apple flowers represent an excellent model in which to characterize how plant microbiomes establish, develop, and correlate with biological processes such as disease progression in an experimentally tractable plant organ.

Author Correction: Evolutionary and expression analysis of CAMTA gene family in Nicotiana tabacum yielded insights into their origin, expansion and stress responses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular breeding approaches for production of disease-resilient commercially important tobacco.

Tobacco is one of the most widely cultivated nonfood cash crops, a source of income, model organism for plant molecular research, a natural pesticide and of pharmaceutical importance. First domesticated in South Americas, the modern-day tobacco (Nicotiana tabacum) is now cultivated in more than 125 countries to generate revenues worth billions of dollars each year. However, the production of this crop is highly threatened by the global presence of devastating infectious agents, which cause huge fiscal loss. These threats have been battled through breeding for acquiring disease resilience in tobacco plants, first, via conventional and now with the use of modern molecular breeding approaches. For efficacy and precision, the characterization of the genetic components underlying disease resistance is the key tool in tobacco for resistance breeding programs. The past few decades have witnessed significant progress in resilience breeding through advanced molecular techniques. The current review discusses history of tobacco breeding since its time of origin till date, highlighting the most widely used techniques and recent advances in molecular research and strategies for resistance breeding. In addition, we narrate the budding possibilities for the future. This review will provide a comprehensive and valuable information for the tobacco growers and researchers to deal with the destructive infectious diseases.

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.

Evolutionary and expression analysis of CAMTA gene family in Nicotiana tabacum yielded insights into their origin, expansion and stress responses.

Calmodulin-binding transcription activators (CAMTAs) represent the novel gene family of transcriptional regulators, which play important biological functions. Though, the first ever plant CAMTA gene was evidenced in Nicotiana tabacum in 2002. But, the systematic identification, origin and function of this gene family has not been performed due to the lack of reference genome information until now. Here, we identified 29 CAMTA genes in four Nicotiana species, including thirteen NtabCAMTAs, six NsylCAMTAs, and five NtomCAMTAs and NbenCAMTAs. These CAMTA families were classified into five phylogenetic groups (I-V), among which, the group-IV CAMTAs probably emerged the earliest. The NtabCAMTA family genes have diverse structures, and are randomly localized on five chromosomes and scaffolds. N. tabacum acquired 11 copies of homolog CAMATA genes from the parental genomes of N. tomentosiformis and N. sylvestris, followed by expansion through polyploidization and duplication. The NtabCAMTA genes were differentially expressed in different plant parts, and showed sensitivity towards different abiotic and biotic stresses. Co-expression network analysis revealed that some NtabCAMTA subunits interact with each other, and co-expressed. The current study is the first report presenting a comprehensive overview of Nicotiana CAMTA families, and opens a new avenue for the improvement of the cultivated tobacco.

Development of a Method to Monitor Gene Expression in Single Bacterial Cells During the Interaction With Plants and Use to Study the Expression of the Type III Secretion System in Single Cells of Dickeya dadantii in Potato.

Dickeya dadantii is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in D. dadantii. Expression of the T3SS is induced in the plant apoplast or in hrp-inducing minimal medium (hrp-MM), and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled in situ observation and quantification of gene expression in single bacterial cells in planta. Using this technique, we observed that the expression of the T3SS genes hrpA and hrpN is restricted to a small proportion of D. dadantii cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS ON and T3SS OFF cells and the expression intensity of T3SS ON cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells in planta.

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations ('Widely Prevalent' clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.

Multi-omics analysis of niche specificity provides new insights into ecological adaptation in bacteria.

Different lifestyles, ranging from a saprophyte to a pathogen, have been reported in bacteria of one species. Here, we performed genome-wide survey of the ecological adaptation in four Burkholderia seminalis strains, distinguished by their origin as part of the saprophytic microbial community of soil or water but also including human and plant pathogens. The results indicated that each strain is separated from the others by increased fitness in medium simulating its original niche corresponding to the difference between strains in metabolic capacities. Furthermore, strain-specific metabolism and niche survival was generally linked with genomic variants and niche-dependent differential expression of the corresponding genes. In particular, the importance of iron, trehalose and d-arabitol utilization was highlighted by the involvement of DNA-methylation and horizontal gene transfer in niche-adapted regulation of the corresponding operons based on the integrated analysis of our multi-omics data. Overall, our results provided insights of niche-specific adaptation in bacteria.

Gene Expression of Type VI Secretion System Associated with Environmental Survival in Acidovorax avenae subsp. avenae by Principle Component Analysis.

Valine glycine repeat G (VgrG) proteins are regarded as one of two effectors of Type VI secretion system (T6SS) which is a complex multi-component secretion system. In this study, potential biological roles of T6SS structural and VgrG genes in a rice bacterial pathogen, Acidovorax avenae subsp. avenae (Aaa) RS-1, were evaluated under seven stress conditions using principle component analysis of gene expression. The results showed that growth of the pathogen was reduced by H2O2 and paraquat-induced oxidative stress, high salt, low temperature, and vgrG mutation, compared to the control. However, pathogen growth was unaffected by co-culture with a rice rhizobacterium Burkholderia seminalis R456. In addition, expression of 14 T6SS structural and eight vgrG genes was significantly changed under seven conditions. Among different stress conditions, high salt, and low temperature showed a higher effect on the expression of T6SS gene compared with host infection and other environmental conditions. As a first report, this study revealed an association of T6SS gene expression of the pathogen with the host infection, gene mutation, and some common environmental stresses. The results of this research can increase understanding of the biological function of T6SS in this economically-important pathogen of rice.

Transcriptome analysis of Acidovorax avenae subsp. avenae cultivated in vivo and co-culture with Burkholderia seminalis.

Response of bacterial pathogen to environmental bacteria and its host is critical for understanding of microbial adaption and pathogenesis. Here, we used RNA-Seq to comprehensively and quantitatively assess the transcriptional response of Acidovorax avenae subsp. avenae strain RS-1 cultivated in vitro, in vivo and in co-culture with rice rhizobacterium Burkholderia seminalis R456. Results revealed a slight response to other bacteria, but a strong response to host. In particular, a large number of virulence associated genes encoding Type I to VI secretion systems, 118 putative non-coding RNAs, and 7 genomic islands (GIs) were differentially expressed in vivo based on comparative genomic and transcriptomic analyses. Furthermore, the loss of virulence for knockout mutants of 11 differentially expressed T6SS genes emphasized the importance of these genes in bacterial pathogenicity. In addition, the reliability of expression data obtained by RNA-Seq was supported by quantitative real-time PCR of the 25 selected T6SS genes. Overall, this study highlighted the role of differentially expressed genes in elucidating bacterial pathogenesis based on combined analysis of RNA-Seq data and knockout of T6SS genes.

Susceptibility of opportunistic Burkholderia glumae to copper surfaces following wet or dry surface contact.

Burkholderia glumae has been proposed to have a potential risk to vulnerable communities. In this work, we investigated the antibacterial activity and mechanism of copper surfaces against multi-drug resistant B. glumae from both patients and rice plants. The susceptibility of B. glumae to copper surfaces was noted by a significant decline in viable bacterial counts, relative to the slight reduction of stainless steel and polyvinylchloride, both of which were used as control surfaces. The mode of action of bacterial killing was determined by examing the mutagenicity, DNA damage, copper ions accumulation, and membrane damage in bacterial cells. The results indicated that the cells exposed to copper surfaces did not cause severe DNA lesions or increase the mutation frequencies, but resulted in a loss of cell membrane integrity within minutes. Furthermore, bacterial cells exposed to copper surfaces accumulated significantly higher amounts of copper compared to control surfaces. Overall, this study showed that metallic copper had strong antibacterial effect against B. glumae by causing DNA and membrane damage, cellular accumulation of copper, and cell death following DNA degradation, which could be utilized to reduce the risk of bacterial contamination and infection.

Genome sequence of the rice pathogen Pseudomonas fuscovaginae CB98818.

Pseudomonas fuscovaginae is a phytopathogenic bacterium causing bacterial sheath brown rot of cereal crops. Here, we present the draft genome sequence of P. fuscovaginae CB98818, originally isolated from a diseased rice plant in China. The draft genome will aid in epidemiological studies, comparative genomics, and quarantine of this broad-host-range pathogen.

Genome sequence of the plant pathogen Pseudomonas syringae pv. panici LMG 2367.

Pseudomonas syringae pv. panici is a phytopathogenic bacterium causing brown stripe disease in economically important crops worldwide. Here, we announce the draft genome sequence of Pseudomonas syringae pv. panici LMG2367 to provide further valuable insights for comparison of the pathovars among species Pseudomonas syringae.