Transcriptomic profiling reveals host-specific evolutionary pathways promoting enhanced fitness in the plant pathogen Ralstonia pseudosolanacearum.

The impact of host diversity on the genotypic and phenotypic evolution of broad-spectrum pathogens is an open issue. Here, we used populations of the plant pathogen Ralstonia pseudosolanacearum that were experimentally evolved on five types of host plants, either belonging to different botanical families or differing in their susceptibility or resistance to the pathogen. We investigated whether changes in transcriptomic profiles, associated with or independent of genetic changes, could occur during the process of host adaptation, and whether transcriptomic reprogramming was dependent on host type. Genomic and transcriptomic variations were established for 31 evolved clones that showed better fitness in their experimental host than the ancestral clone. Few genomic polymorphisms were detected in these clones, but significant transcriptomic variations were observed, with a large number of differentially expressed genes (DEGs). In a very clear way, a group of genes belonging to the network of regulation of the bacterial virulence such as efpR, efpH or hrpB, among others, were deregulated in several independent evolutionary lineages and appeared to play a key role in the transcriptomic rewiring observed in evolved clones. A double hierarchical clustering based on the 400 top DEGs for each clone revealed 2 major patterns of gene deregulation that depend on host genotype, but not on host susceptibility or resistance to the pathogen. This work therefore highlights the existence of two major evolutionary paths that result in a significant reorganization of gene expression during adaptive evolution and underscore clusters of co-regulated genes associated with bacterial adaptation on different host lines.

Ralstonia solanacearum: An Arsenal of Virulence Strategies and Prospects for Resistance.

The group of strains constituting the Ralstonia solanacearum species complex (RSSC) is a prominent model for the study of plant-pathogenic bacteria because of its impact on agriculture, owing to its wide host range, worldwide distribution, and long persistence in the environment. RSSC strains have led to numerous studies aimed at deciphering the molecular bases of virulence, and many biological functions and mechanisms have been described to contribute to host infection and pathogenesis. In this review, we put into perspective recent advances in our understanding of virulence in RSSC strains, both in terms of the inventory of functions that participate in this process and their evolutionary dynamics. We also present the different strategies that have been developed to combat these pathogenic strains through biological control, antimicrobial agents, plant genetics, or microbiota engineering.

Insights into the metabolic specificities of pathogenic strains from the Ralstonia solanacearum species complex.

All the strains grouped under the species Ralstonia solanacearum represent a species complex responsible for many diseases on agricultural crops throughout the world. The strains have different lifestyles and host range. Here, we investigated whether specific metabolic pathways contribute to strain diversification. To this end, we carried out systematic comparisons on 11 strains representing the diversity of the species complex. We reconstructed the metabolic network of each strain from its genome sequence and looked for the metabolic pathways differentiating the different reconstructed networks and, by extension, the different strains. Finally, we conducted an experimental validation by determining the metabolic profile of each strain with the Biolog technology. Results revealed that the metabolism is conserved between strains, with a core metabolism composed of 82% of the pan-reactome. The three species composing the species complex could be distinguished according to the presence/absence of some metabolic pathways, in particular, one involving salicylic acid degradation. Phenotypic assays revealed that the trophic preferences on organic acids and several amino acids such as glutamine, glutamate, aspartate, and asparagine are conserved between strains. Finally, we generated mutants lacking the quorum-sensing-dependent regulator PhcA in four diverse strains, and we showed that the phcA-dependent trade-off between growth and production of virulence factors is conserved across the R. solanacearum species complex. IMPORTANCE Ralstonia solanacearum is one of the most important threats to plant health worldwide, causing disease on a very large range of agricultural crops such as tomato or potato. Behind the R. solanacearum name are hundreds of strains with different host range and lifestyle, classified into three species. Studying the differences between strains allows to better apprehend the biology of the pathogens and the specificity of some strains. None of the published genomic comparative studies have focused on the metabolism of the strains so far. We developed a new bioinformatic pipeline to build high-quality metabolic networks and used a combination of metabolic modeling and high-throughput phenotypic Biolog microplates to look for the metabolic differences between 11 strains across the three species. Our study revealed that genes encoding enzymes are overall conserved, with few variations between strains. However, more variations were observed when considering substrate usage. These variations probably result from regulation rather than the presence or absence of enzymes in the genome.

An atypical NLR gene confers bacterial wilt susceptibility in Arabidopsis.

Quantitative disease resistance (QDR) remains the most prevalent form of plant resistance in crop fields and wild habitats. Genome-wide association studies (GWAS) have proved to be successful in deciphering the quantitative genetic basis of complex traits such as QDR. To unravel the genetics of QDR to the devastating worldwide bacterial pathogen Ralstonia solanacearum, we performed a GWAS by challenging a highly polymorphic local mapping population of Arabidopsis thaliana with four R. solanacearum type III effector (T3E) mutants, identified as key pathogenicity determinants after a first screen on an A. thaliana core collection of 25 accessions. Although most quantitative trait loci (QTLs) were highly specific to the identity of the T3E mutant (ripAC, ripAG, ripAQ, and ripU), we finely mapped a common QTL located on a cluster of nucleotide-binding domain and leucine-rich repeat (NLR) genes that exhibited structural variation. We functionally validated one of these NLRs as a susceptibility factor in response to R. solanacearum, named it Bacterial Wilt Susceptibility 1 (BWS1), and cloned two alleles that conferred contrasting levels of QDR. Further characterization indicated that expression of BWS1 leads to suppression of immunity triggered by different R. solanacearum effectors. In addition, we showed a direct interaction between BWS1 and RipAC T3E, and BWS1 and SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1b), the latter interaction being suppressed by RipAC. Together, our results highlight a putative role for BWS1 as a quantitative susceptibility factor directly targeted by the T3E RipAC, mediating negative regulation of the SGT1-dependent immune response.

Trophic preferences of the pathogen Ralstonia solanacearum and consequences on its growth in xylem sap.

Ralstonia solanacearum is one of the most destructive pathogens worldwide. In the last 30 years, the molecular mechanisms at the origin of R. solanacearum pathogenicity have been studied in depth. However, the nutrition status of the pathogen once inside the plant has been poorly investigated. Yet, the pathogen needs substrates to sustain a fast-enough growth, maintain its virulence and subvert the host immunity. This study aimed to explore in-depth the xylem environment where the pathogen is abundant, and its trophic preferences. First, we determined the composition of tomato xylem sap, where fast multiplication of the pathogen occurs. Then, kinetic growth on single and mixtures of carbon sources in relation to this environment was performed to fully quantify growth. Finally, we calculated the concentration of available metabolites in the xylem sap flux to assess how much it can support bacterial growth in planta. Overall, the study underlines the adaptation of R. solanacearum to the xylem environment and the fact that the pathogen assimilates several substrates at the same time in media composed of several carbon sources. It also provides metrics on key physiological parameters governing the growth of this major pathogen, which will be instrumental in the future to better understand its metabolic behavior during infection.

Study of natural diversity in response to a key pathogenicity regulator of Ralstonia solanacearum reveals new susceptibility genes in Arabidopsis thaliana.

Ralstonia solanacearum gram-negative phytopathogenic bacterium exerts its virulence through a type III secretion system (T3SS) that translocates type III effectors (T3Es) directly into the host cells. T3E secretion is finely controlled at the posttranslational level by helper proteins, T3SS control proteins, and type III chaperones. The HpaP protein, one of the type III secretion substrate specificity switch (T3S4) proteins, was previously highlighted as a virulence factor on Arabidopsis thaliana Col-0 accession. In this study, we set up a genome-wide association analysis to explore the natural diversity of response to the hpaP mutant of two A. thaliana mapping populations: a worldwide collection and a local population. Quantitative genetic variation revealed different genetic architectures in both mapping populations, with a global delayed response to the hpaP mutant compared to the GMI1000 wild-type strain. We have identified several quantitative trait loci (QTLs) associated with the hpaP mutant inoculation. The genes underlying these QTLs are involved in different and specific biological processes, some of which were demonstrated important for R. solanacearum virulence. We focused our study on four candidate genes, RKL1, IRE3, RACK1B, and PEX3, identified using the worldwide collection, and validated three of them as susceptibility factors. Our findings demonstrate that the study of the natural diversity of plant response to a R. solanacearum mutant in a key regulator of virulence is an original and powerful strategy to identify genes directly or indirectly targeted by the pathogen.

A multi-organ metabolic model of tomato predicts plant responses to nutritional and genetic perturbations.

Predicting and understanding plant responses to perturbations require integrating the interactions between nutritional sources, genes, cell metabolism, and physiology in the same model. This can be achieved using metabolic modeling calibrated by experimental data. In this study, we developed a multi-organ metabolic model of a tomato (Solanum lycopersicum) plant during vegetative growth, named Virtual Young TOmato Plant (VYTOP) that combines genome-scale metabolic models of leaf, stem and root and integrates experimental data acquired from metabolomics and high-throughput phenotyping of tomato plants. It is composed of 6,689 reactions and 6,326 metabolites. We validated VYTOP predictions on five independent use cases. The model correctly predicted that glutamine is the main organic nutrient of xylem sap. The model estimated quantitatively how stem photosynthetic contribution impacts exchanges between the different organs. The model was also able to predict how nitrogen limitation affects vegetative growth and the metabolic behavior of transgenic tomato lines with altered expression of core metabolic enzymes. The integration of different components, such as a metabolic model, physiological constraints, and experimental data, generates a powerful predictive tool to study plant behavior, which will be useful for several other applications, such as plant metabolic engineering or plant nutrition.

Polyamines: double agents in disease and plant immunity.

Polyamines (PAs) are ubiquitous amine molecules found in all living organisms. In plants, beside their role in signaling and protection against abiotic stresses, there is increasing evidence that PAs have a major role in the interaction between plants and pathogens. Plant PAs are involved in immunity against pathogens, notably by amplifying pattern-triggered immunity (PTI) responses through the production of reactive oxygen species (ROS). In response, pathogens use phytotoxins and effectors to manipulate the levels of PAs in the plant, most likely to their own benefit. It also appears that pathogenic microorganisms produce PAs during infection, sometimes in large quantities. This may reflect different infectious strategies based on the selective exploitation of these molecules and the functions they perform in the cell.

Unravelling physiological signatures of tomato bacterial wilt and xylem metabolites exploited by Ralstonia solanacearum.

The plant pathogen Ralstonia solanacearum uses plant resources to intensely proliferate in xylem vessels and provoke plant wilting. We combined automatic phenotyping and tissue/xylem quantitative metabolomics of infected tomato plants to decipher the dynamics of bacterial wilt. Daily acquisition of physiological parameters such as transpiration and growth were performed. Measurements allowed us to identify a tipping point in bacterial wilt dynamics. At this tipping point, the reached bacterial density brutally disrupts plant physiology and rapidly induces its death. We compared the metabolic and physiological signatures of the infection with drought stress, and found that similar changes occur. Quantitative dynamics of xylem content enabled us to identify glutamine (and asparagine) as primary resources R. solanacearum consumed during its colonization phase. An abundant production of putrescine was also observed during the infection process and was strongly correlated with in planta bacterial growth. Dynamic profiling of xylem metabolites confirmed that glutamine is the favoured substrate of R. solanacearum. On the other hand, a triple mutant strain unable to metabolize glucose, sucrose and fructose appears to be only weakly reduced for in planta growth and pathogenicity.

Convergent Rewiring of the Virulence Regulatory Network Promotes Adaptation of Ralstonia solanacearum on Resistant Tomato.

The evolutionary and adaptive potential of a pathogen is a key determinant for successful host colonization and proliferation but remains poorly known for most of the pathogens. Here, we used experimental evolution combined with phenotyping, genomics, and transcriptomics to estimate the adaptive potential of the bacterial plant pathogen Ralstonia solanacearum to overcome the quantitative resistance of the tomato cultivar Hawaii 7996. After serial passaging over 300 generations, we observed pathogen adaptation to within-plant environment of the resistant cultivar but no plant resistance breakdown. Genomic sequence analysis of the adapted clones revealed few genetic alterations, but we provide evidence that all but one were gain of function mutations. Transcriptomic analyses revealed that even if different adaptive events occurred in independently evolved clones, there is convergence toward a global rewiring of the virulence regulatory network as evidenced by largely overlapping gene expression profiles. A subset of four transcription regulators, including HrpB, the activator of the type 3 secretion system regulon and EfpR, a global regulator of virulence and metabolic functions, emerged as key nodes of this regulatory network that are frequently targeted to redirect the pathogen's physiology and improve its fitness in adverse conditions. Significant transcriptomic variations were also detected in evolved clones showing no genomic polymorphism, suggesting that epigenetic modifications regulate expression of some of the virulence network components and play a major role in adaptation as well.

Genome-Scale Investigation of the Metabolic Determinants Generating Bacterial Fastidious Growth.

High proliferation rate and robustness are vital characteristics of bacterial pathogens that successfully colonize their hosts. The observation of drastically slow growth in some pathogens is thus paradoxical and remains unexplained. In this study, we sought to understand the slow (fastidious) growth of the plant pathogen Xylella fastidiosa Using genome-scale metabolic network reconstruction, modeling, and experimental validation, we explored its metabolic capabilities. Despite genome reduction and slow growth, the pathogen's metabolic network is complete but strikingly minimalist and lacking in robustness. Most alternative reactions were missing, especially those favoring fast growth, and were replaced by less efficient paths. We also found that the production of some virulence factors imposes a heavy burden on growth. Interestingly, some specific determinants of fastidious growth were also found in other slow-growing pathogens, enriching the view that these metabolic peculiarities are a pathogenicity strategy to remain at a low population level.IMPORTANCE Xylella fastidiosa is one of the most important threats to plant health worldwide, causing disease in the Americas on a range of agricultural crops and trees, and recently associated with a critical epidemic affecting olive trees in Europe. A main challenge for the detection of the pathogen and the development of physiological studies is its fastidious growth, as the generation time can vary from 10 to 100 h for some strains. This physiological peculiarity is shared with several human pathogens and is poorly understood. We performed an analysis of the metabolic capabilities of X. fastidiosa through a genome-scale metabolic model of the bacterium. This model was reconstructed and manually curated using experiments and bibliographical evidence. Our study revealed that fastidious growth most probably results from different metabolic specificities such as the absence of highly efficient enzymes or a global inefficiency in virulence factor production. These results support the idea that the fragility of the metabolic network may have been shaped during evolution to lead to the self-limiting behavior of X. fastidiosa.

HpaP Sequesters HrpJ, an Essential Component of Ralstonia solanacearum Virulence That Triggers Necrosis in Arabidopsis.

The Gram-negative bacterium Ralstonia solanacearum, the causal agent of bacterial wilt, is a worldwide major crop pathogen whose virulence strongly relies on a type III secretion system (T3SS). This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. To date, very few data are available in plant-pathogenic bacteria concerning the role played by type III secretion (T3S) regulators at the posttranslational level. We have demonstrated that HpaP, a putative T3S substrate specificity switch protein of R. solanacearum, controls T3E secretion. To better understand the role of HpaP on T3S control, we analyzed the secretomes of the GMI1000 wild-type strain as well as the hpaP mutant using a mass spectrometry experiment (liquid chromatography tandem mass spectrometry). The secretomes of both strains appeared to be very similar and highlighted the modulation of the secretion of few type III substrates. Interestingly, only one type III-associated protein, HrpJ, was identified as specifically secreted by the hpaP mutant. HrpJ appeared to be an essential component of the T3SS, essential for T3S and pathogenicity. We further showed that HrpJ is specifically translocated in planta by the hpaP mutant and that HrpJ can physically interact with HpaP. Moreover, confocal microscopy experiments demonstrated a cytoplasmic localization for HrpJ once in planta. When injected into Arabidopsis thaliana leaves, HrpJ is able to trigger a necrosis on 16 natural accessions. A genome-wide association mapping revealed a major association peak with 12 highly significant single-nucleotide polymorphisms located on a plant acyl-transferase.

Trehalose Synthesis Contributes to Osmotic Stress Tolerance and Virulence of the Bacterial Wilt Pathogen Ralstonia solanacearum.

The xylem-dwelling plant pathogen Ralstonia solanacearum changes the chemical composition of host xylem sap during bacterial wilt disease. The disaccharide trehalose, implicated in stress tolerance across all kingdoms of life, is enriched in sap from R. solanacearum-infected tomato plants. Trehalose in xylem sap could be synthesized by the bacterium, the plant, or both. To investigate the source and role of trehalose metabolism during wilt disease, we evaluated the effects of deleting the three trehalose synthesis pathways in the pathogen: TreYZ, TreS, and OtsAB, as well as its sole trehalase, TreA. A quadruple treY/treS/otsA/treA mutant produced 30-fold less intracellular trehalose than the wild-type strain missing the trehalase enzyme. This trehalose-nonproducing mutant had reduced tolerance to osmotic stress, which the bacterium likely experiences in plant xylem vessels. Following naturalistic soil-soak inoculation of tomato plants, this triple mutant did not cause disease as well as wild-type R. solanacearum. Further, the wild-type strain out-competed the trehalose-nonproducing mutant by over 600-fold when tomato plants were coinoculated with both strains, showing that trehalose biosynthesis helps R. solanacearum overcome environmental stresses during infection. An otsA (trehalose-6-phosphate synthase) single mutant behaved similarly to ΔtreY/treS/otsA in all experimental settings, suggesting that the OtsAB pathway is the dominant trehalose synthesis pathway in R. solanacearum.

Pangenomic type III effector database of the plant pathogenic Ralstonia spp.

BACKGROUND: The bacterial plant pathogenic Ralstonia species belong to the beta-proteobacteria class and are soil-borne pathogens causing vascular bacterial wilt disease, affecting a wide range of plant hosts. These bacteria form a heterogeneous group considered as a "species complex" gathering three newly defined species. Like many other Gram negative plant pathogens, Ralstonia pathogenicity relies on a type III secretion system, enabling bacteria to secrete/inject a large repertoire of type III effectors into their plant host cells. Type III-secreted effectors (T3Es) are thought to participate in generating a favorable environment for the pathogen (countering plant immunity and modifying the host metabolism and physiology). METHODS: Expert genome annotation, followed by specific type III-dependent secretion, allowed us to improve our Hidden-Markov-Model and Blast profiles for the prediction of type III effectors. RESULTS: We curated the T3E repertoires of 12 plant pathogenic Ralstonia strains, representing a total of 12 strains spread over the different groups of the species complex. This generated a pangenome repertoire of 102 T3E genes and 16 hypothetical T3E genes. Using this database, we scanned for the presence of T3Es in the 155 available genomes representing 140 distinct plant pathogenic Ralstonia strains isolated from different host plants in different areas of the globe. All this information is presented in a searchable database. A presence/absence analysis, modulated by a strain sequence/gene annotation quality score, enabled us to redefine core and accessory T3E repertoires.

Spontaneous mutations in a regulatory gene induce phenotypic heterogeneity and adaptation of Ralstonia solanacearum to changing environments.

An evolution experiment with the bacterial plant pathogen Ralstonia solanacearum revealed that several adaptive mutations conferring enhanced fitness in plants arose in the efpR gene encoding a regulator of virulence and metabolic functions. In this study, we found that an efpR mutant systematically displays colonies with two morphotypes: the type S ('smooth', similar to the wild type) and the type EV ('efpR variant'). We demonstrated that the efpH gene, a homologue of efpR, plays a key role in the control of phenotypic heterogeneity, the ΔefpR-ΔefpH double mutant being stably locked into the EV type. Using mixed infection assays, we demonstrated that the type EV is metabolically more proficient than the type S and displays fitness gain in specific environments, whereas the type S has a better fitness into the plant environment. We provide evidence that this efpR-dependent phenotypic heterogeneity is a general feature of strains of the R. solanacearum species complex and could occur in natural conditions. This study highlights the potential role of phenotypic heterogeneity in this plant pathogen as an adaptive trait to changing environments.

The eggplant AG91-25 recognizes the Type III-secreted effector RipAX2 to trigger resistance to bacterial wilt (Ralstonia solanacearum species complex).

To deploy durable plant resistance, we must understand its underlying molecular mechanisms. Type III effectors (T3Es) and their recognition play a central role in the interaction between bacterial pathogens and crops. We demonstrate that the Ralstonia solanacearum species complex (RSSC) T3E ripAX2 triggers specific resistance in eggplant AG91-25, which carries the major resistance locus EBWR9. The eggplant accession AG91-25 is resistant to the wild-type R. pseudosolanacearum strain GMI1000, whereas a ripAX2 defective mutant of this strain can cause wilt. Notably, the addition of ripAX2 from GMI1000 to PSS4 suppresses wilt development, demonstrating that RipAX2 is an elicitor of AG91-25 resistance. RipAX2 has been shown previously to induce effector-triggered immunity (ETI) in the wild relative eggplant Solanum torvum, and its putative zinc (Zn)-binding motif (HELIH) is critical for ETI. We show that, in our model, the HELIH motif is not necessary for ETI on AG91-25 eggplant. The ripAX2 gene was present in 68.1% of 91 screened RSSC strains, but in only 31.1% of a 74-genome collection comprising R. solanacearum and R. syzygii strains. Overall, it is preferentially associated with R. pseudosolanacearum phylotype I. RipAX2GMI1000 appears to be the dominant allele, prevalent in both R. pseudosolanacearum and R. solanacearum, suggesting that the deployment of AG91-25 resistance could control efficiently bacterial wilt in the Asian, African and American tropics. This study advances the understanding of the interaction between RipAX2 and the resistance genes at the EBWR9 locus, and paves the way for both functional genetics and evolutionary analyses.

Comparative transcriptomic studies identify specific expression patterns of virulence factors under the control of the master regulator PhcA in the Ralstonia solanacearum species complex.

The global regulator PhcA controls numerous traits associated to virulence and bacterial proliferation in strains of the plant pathogen Ralstonia solanacearum species complex. Here, we conducted a genome-wide RNA sequencing study of the GMI1000 wild-type strain and a derived phcA mutant grown in complete medium. The PhcA regulon we identified is the largest regulon described to date in the R. solanacearum species complex with 1581 regulated genes, representing about 30% of the bacterial genome. Among these genes, 166 transcription regulators were identified including known regulators controlling major cellular functions such as the Type 3 secretion system and 27 novel regulators that were not identified in previous transcriptomic studies. This study highlights that PhcA controls other functions beside pathogenicity stricto sensu which participate to the global cell homeostasis (metabolism, energy storage). We then compared the PhcA regulon identified in complete medium to the recently published PhcA regulon obtained in planta. This comparison of the set of GMI1000 genes subjected to PhcA regulation in both conditions revealed 383 common genes. Among them, 326 (85%) had a similar PhcA dependent regulation pattern in complete medium and in planta, and 57 (15%) displayed an opposite regulation pattern. A large majority of the genes repressed by PhcA in complete medium but activated in planta belong to the HrpG-HrpB regulon, which represents a set of key genes required for R. solanacearum pathogenesis. This latter class of genes appears to be specifically induced by PhcA in the plant environment whereas PhcA represses their expression in complete medium. The large set of direct and indirect targets identified in this study will contribute to enrich our knowledge of the intricate regulatory network coordinating the expression of virulence and metabolic functions in the model plant pathogen R. solanacearum.

Control of primary metabolism by a virulence regulatory network promotes robustness in a plant pathogen.

Robustness is a key system-level property of living organisms to maintain their functions while tolerating perturbations. We investigate here how a regulatory network controlling multiple virulence factors impacts phenotypic robustness of a bacterial plant pathogen. We reconstruct a cell-scale model of Ralstonia solanacearum connecting a genome-scale metabolic network, a virulence macromolecule network, and a virulence regulatory network, which includes 63 regulatory components. We develop in silico methods to quantify phenotypic robustness under a broad set of conditions in high-throughput simulation analyses. This approach reveals that the virulence regulatory network exerts a control of the primary metabolism to promote robustness upon infection. The virulence regulatory network plugs into the primary metabolism mainly through the control of genes likely acquired via horizontal gene transfer, which results in a functional overlay with ancestral genes. These results support the view that robustness may be a selected trait that promotes pathogenic fitness upon infection.

Introduction of Genetic Material in Ralstonia solanacearum Through Natural Transformation and Conjugation.

Ralstonia solanacearum is a soil-borne plant pathogen, responsible of the bacterial wilt disease. Its unusual wide host range (more than 250 plant species), aggressiveness, and broad geographic distribution have made of this bacterium the main plant pathogenic model in the beta-Proteobacteria class. Many R. solanacearum strains have the ability to internalize exogenous DNA through natural transformation. This property is widely used in reverse genetics studies to create mutants or reporter gene constructs, in the aim to study the molecular bases of pathogenesis of this bacterium. In this chapter, we describe three in vitro methods (natural transformation, electrotransformation, and conjugation) commonly used to produce recombinant R. solanacearum cells after introduction of exogenous DNA.