Two species with a peculiar evolution within the genus Pectobacterium suggest adaptation to a new environmental niche.

Historically, research on Soft Rot Pectobacteriacea (SRP) has focused on economically important crops and ornamentals and knowledge of these bacteria outside the plant context remains poorly investigated. Recently, two closely related species Pectobacterium aquaticum and Pectobacterium quasiaquaticum were isolated from water and have not been isolated from any plant yet. To identify the distinctive characteristics of these two species, we performed a comparative genomic analysis of 80 genomes representing 19 Pectobacterium species and performed an evolutionary reconstruction. Both water species underwent a reduction in genome size associated with a high pseudogene content. A high gene loss was predicted at the emergence of both species. Among the 199 gene families missing from both P. aquaticum and P. quasiaquaticum genomes but present in at least 80% of other Pectobacterium genomes, COG analysis identified many genes involved in nutrient transport systems. In addition, many type II secreted proteins were also missing in both species. Phenotypic analysis revealed that both species had reduced pectinolytic activity, a biofilm formation defect, were highly motile and had reduced virulence on several plants. These genomic and phenotypic data suggest that the ecological niche of P. aquaticum and P. quasiaquaticum may differ from that of other Pectobacterium species.

Genetic and Phenotypic Study of the Pectobacterium versatile Beta-Lactamase, the Enzyme Most Similar to the Plasmid-Encoded TEM-1.

Genus Pectobacterium bacteria include important agricultural pathogens. Pectobacterium versatile isolates contain a chromosome-borne beta-lactamase, PEC-1. This enzyme is the closest relative of TEM-1, a plasmid-borne beta-lactamase widespread in the Enterobacterales. We performed bioinformatics and phenotypic analyses to investigate the genetic and phenotypic features of PEC-1 and its frequency and ability to spread within genus Pectobacterium. We also compared the characteristics of PEC-1 and TEM-1 and evaluated the likelihood of transfer. We found that blaPEC-1 was present principally in a small number of genetic environments in P. versatile. Identical blaPEC-1 genetic environments were present in closely related species, consistent with the high frequency of genetic exchange within the genus Pectobacterium. Despite the similarities between PEC-1 and TEM-1, their genetic environments displayed no significant identity, suggesting an absence of recent transfer. Phenotypic analyses on clonal constructs revealed similar hydrolysis spectra. Our results suggest that P. versatile is the main reservoir of PEC-1, which seems to transfer to closely related species. The genetic distance between PEC-1 and TEM-1, and the lack of conserved elements in their genetic environments, suggest that any transfer that may have occurred must have taken place well before the antibiotic era. IMPORTANCE This study aimed to compare the chromosomal beta-lactamase from Pectobacterium versatile, PEC-1, with the well-known and globally distributed TEM-1 in terms of genetic and functional properties. Despite the similarities between the enzymes, we obtained no definitive proof of gene transfer for the emergence of blaPEC-1 from blaTEM-1. Indeed, given the limited degree of sequence identity and the absence of a common genetic environment, it seems unlikely that any transfer of this gene has occurred recently. However, although blaPEC-1 was found mostly in one specific clade of the P. versatile species, certain isolates from other closely related species, such as Pectobacterium brasiliense and Pectobacterium polaris, may also carry this gene inserted into common genetic environments. This observation suggests that genetic exchanges are frequent, accounting for the diffusion of blaPEC-1 between isolates from different Pectobacterium species and, potentially, to exogenous mobile genetic elements.

The Diversity and Abundance of Soft Rot Pectobacteriaceae Along the Durance River Stream in the Southeast of France Revealed by Multiple Seasonal Surveys.

Although irrigation water is frequently assessed for the presence of plant pathogens, large spatial and temporal surveys that provide clues on the diversity and circulation of pathogens are missing. We evaluate the diversity of soft rot Pectobacteriaceae (SRP) of the genera Dickeya and Pectobacterium over 2 years in a temperate, mixed-use watershed. The abundance of isolated strains correlates with the agricultural gradient along the watershed with a positive correlation found with temperature, nitrate, and dissolved organic carbon water concentration. We characterized 582 strains by amplification and sequencing of the gapA gene. Multilocus sequence analysis, performed with three housekeeping genes for 99 strains, and core genome analysis of 38 sequenced strains, confirmed for all the strains but one, the taxonomic assignation obtained with the sole gapA sequence. Pectobacterium spp. (549 isolates) were far more abundant than Dickeya spp. (33 isolates). Dickeya spp. were only observed in the lower part of the river when water temperature was >19°C, and we experimentally confirmed a decreased fitness of several Dickeya spp. at 8°C in river water. D. oryzae dominates the Dickeya spp. and P. versatile and P. aquaticum dominate the Pectobacterium spp., but their repartition along the watershed was different, with P. versatile being the only species regularly recovered all along the watershed. Excepting P. versatile, the Dickeya and Pectobacterium spp. responsible for disease outbreak on crops were less abundant or rarely detected. This work sheds light on the various ecological behaviors of different SRP types in stream water and indicates that SRP occupation is geographically structured.

Pectobacterium quasiaquaticum sp. nov., isolated from waterways.

Through this study, we established the taxonomic status of seven strains belonging to the genus Pectobacterium (A477-S1-J17T, A398-S21-F17, A535-S3-A17, A411-S4-F17, A113-S21-F16, FL63-S17 and FL60-S17) collected from four different river streams and an artificial lake in south-east France between 2016 and 2017. Ecological surveys in rivers and lakes pointed out different repartition of strains belonging to this clade compared to the closest species, Pectobacterium aquaticum. The main phenotypic difference observed between these strains and the P. aquaticum type strain was strongly impaired growth with rhamnose as the sole carbon source. This correlates with three different forms of pseudogenization of the l-rhamnose/proton symporter gene rhaT in the genomes of strains belonging to this clade. Phylogenetic analysis using gapA gene sequences and multi locus sequence analysis of the core genome showed that these strains formed a distinct clade within the genus Pectobacterium closely related to P. aquaticum. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values showed a clear discontinuity between the new clade and P. aquaticum. However, the calculated values are potentially consistent with either splitting or merging of this new clade with P. aquaticum. In support of the split, ANI coverages were higher within this new clade than between this new clade and P. aquaticum. The split is also consistent with the range of observed ANI or dDDH values that currently separate several accepted species within the genus Pectobacterium. On the basis of these data,strains A477-S1-J17T, A398-S21-F17, A535-S3-A17, A411-S4-F17, A113-S21-F16, FL63-S17 and FL60-S17 represent a novel species of the genus Pectobacterium, for which the name Pectobacterium quasiaquaticum sp. nov. is proposed. The type strain is A477-S1-J17T (=CFBP 8805T=LMG 32181T).

Updated Taxonomy of Pectobacterium Genus in the CIRM-CFBP Bacterial Collection: When Newly Described Species Reveal "Old" Endemic Population.

Bacterial collections are invaluable tools for microbiologists. However, their practical use is compromised by imprecise taxonomical assignation of bacterial strains. This is particularly true for soft rotting plant pathogens of the Pectobacterium genus. We analysed the taxonomic status of 265 Pectobacterium strains deposited at CIRM-CFBP collection from 1944 to 2020. This collection gathered Pectobacterium strains isolated in 27 countries from 32 plant species representing 17 botanical families or from nonhost environments. The MLSA approach completed by genomic analysis of 15 strains was performed to update the taxonomic status of these 265 strains. The results showed that the CIRM-CFBP Pectobacterium collection harboured at least one strain of each species, with the exception of P. polonicum. Yet, seven strains could not be assigned to any of the described species and may represent at least two new species. Surprisingly, P. versatile, recently described in 2019, is the most prevalent species among CIRM-CFBP strains. An analysis of P. versatile strains revealed that this species is pandemic and isolated from various host plants and environments. At the opposite, other species gathered strains isolated from only one botanical family or exclusively from a freshwater environment. Our work also revealed new host plants for several Pectobacterium spp.

Comparison of Environmental and Culture-Derived Bacterial Communities through 16S Metabarcoding: A Powerful Tool to Assess Media Selectivity and Detect Rare Taxa.

To compare environmental and culture-derived microbial communities, we performed 16S metabarcoding of uncultured samples and their culture-derived bacterial lawns. Microbial communities were obtained from freshwater river samples representative of an anthropization gradient along a river stream. Their culture-derived bacterial lawns were obtained by growing aliquots of the samples on a broad range medium and on two different semi-selective media. The V3-V4 16S rRNA region was amplified and sequenced. The bacterial diversity of water samples decreased from the upper to lower stream sampling sites and, as expected, these differences were mostly suppressed by the culture step. Overall, the diversity of cultured-derived bacterial communities reflected selectivity of each tested medium. Comparison of treatments indicated that the culture selected both detected and rare undetected environmental species. Accurate detection of rare environmental bacteria of the Pectobacterium genus by 16S metabarcoding of the culture lawn was demonstrated. Interestingly, for abundant taxa, such as those of the Pseudomonas genus, the culture/environment ratio varied between sampled sites, indicating the difficulty of comparing cultured-derived taxa abundance between environmental sites. Finally, our study also highlighted media specificity and complementarity: bacterial communities grown on the two selective media, while selecting a small set of specific species, were mostly a subset of the bacterial community observed on the broad range medium.

Elevation of Pectobacterium carotovorum subsp. odoriferum to species level as Pectobacterium odoriferum sp. nov., proposal of Pectobacterium brasiliense sp. nov. and Pectobacterium actinidiae sp. nov., emended description of Pectobacterium carotovorum and description of Pectobacterium versatile sp. nov., isolated from streams and symptoms on diverse plants.

The Pectobacteriumcarotovorum species corresponds to a complex, including two subspecies with validly published names, two proposed subspecies and two new species, Pectobacterium polaris and Pectobacterium aquaticum. Recent studies suggested that this complex needed revision. We examined the taxonomic status of 144 Pectobacterium strains isolated from a wide range of plant species, various geographical origins and waterways. Sequences of the leuS, dnaX and recA housekeeping genes clustered 114 of these Pectobacterium strains together within a not yet described clade. We sequenced eight strains of this clade and analysed them together with the 102 Pectobacterium genomes available in the NCBI database. Phylogenetic analysis, average nucleotide identity calculation and in silico DNA-DNA hybridization allowed us to differentiate seven clades. This led us to propose the elevation of Pectobacterium carotovorumsubsp. odoriferum to species level as Pectobacteriumodoriferum sp. nov. (type strain CFBP 1878T=LMG 5863T=NCPPB 3839T=ICMP 11533T), the proposal of Pectobacteriumactinidiae sp. nov. (type strain KKH3=LMG 26003 T=KCTC 23131T) and Pectobacteriumbrasiliense sp. nov. (type strain CFBP 6617T= LMG 21371T=NCPPB 4609T), to emend the description of Pectobacterium carotovorum (type strain CFBP 2046T=LMG 2404T=NCPPB 312T=ICMP 5702T), and to propose a novel species, Pectobacterium versatile sp. nov (type strain CFBP6051T= NCPPB 3387T=ICMP 9168T) which includes the strains previously described as 'Candidatus Pectobacterium maceratum'. Phenotypic analysis performed using Biolog GENIII plates on eight strains of P. versatile sp. nov. and related strains completed our analysis.

Pectobacterium aquaticum sp. nov., isolated from waterways.

This work aimed to establish the taxonomic status of six strains (A212-S19-A16T, A127-S21-F16, A105-S21-F16, A104-S21-F16, A101-S19-F16 and A35-S23-M15) isolated from three different waterways in 2015 and 2016 in south-east France. Amplification and sequencing of the gapA housekeeping gene clustered these six strains together inside the genus Pectobacterium outside of already described or proposed Pectobacterium species and supspecies. Phenotypic analysis, using GENIII Biolog plates performed with strains A212-S19-A16T, A105-S21-F16, A101-S19-F16 and the closely related Pectobacterium polaris(CFBP 1403), Pectobacterium carotovorum subsp. odoriferum (CFBP 1878T), 'Pectobacteriumcarotovorum subsp. actinidiae' (CFBP 7370), Pectobacterium carotovorum subsp. carotovorum (CFBP 2046T), 'Pectobacterium carotovorum subsp. brasiliense' (CFBP 6617) or the most distantly related Pectobacteriumaroidearum (CFBP 8168T) failed to identify specific compounds metabolized by these three strains, but weak activity was specifically observed at pH 5 with these three strains. Illumina sequencing was used to sequence these six strains. Based on phylogenetic data, average nucleotide identity values and in silico DNA-DNA hybridization results, strains A212-S19-A16T, A127-S21-F16, A105-S21-F16, A101-S19-F16, A35-S23-M15 and A104-S21-F16 are suggested to represent a novel species of the genus Pectobacterium, for which the name Pectobacterium aquaticum sp. nov. is proposed. The type strain is A212-S19-A16 T (=CFBP 8637T=NCPPB 4640T).

Draft genomes of "Pectobacterium peruviense" strains isolated from fresh water in France.

Bacteria belonging to the genus Pectobacterium are responsible for soft rot disease on a wide range of cultivated crops. The "Pectobacterium peruviense" specie, recently proposed inside the Pectobacterium genus, gathers strains isolated from potato tubers cultivated in Peru at high altitude. Here we report the draft genome sequence of two strains belonging to "P. peruviense" isolated from river water in France indicating that the geographic distribution of this specie is likely to be larger than previously anticipated. We compared these genomes with the one published from the "P. peruviense" specie type strain isolated in Peru.

Insights into the Planktothrix genus: Genomic and metabolic comparison of benthic and planktic strains.

Planktothrix is a dominant cyanobacterial genus forming toxic blooms in temperate freshwater ecosystems. We sequenced the genome of planktic and non planktic Planktothrix strains to better represent this genus diversity and life style at the genomic level. Benthic and biphasic strains are rooting the Planktothrix phylogenetic tree and widely expand the pangenome of this genus. We further investigated in silico the genetic potential dedicated to gas vesicles production, nitrogen fixation as well as natural product synthesis and conducted complementary experimental tests by cell culture, microscopy and mass spectrometry. Significant differences for the investigated features could be evidenced between strains of different life styles. The benthic Planktothrix strains showed unexpected characteristics such as buoyancy, nitrogen fixation capacity and unique natural product features. In comparison with Microcystis, another dominant toxic bloom-forming genus in freshwater ecosystem, different evolutionary strategies were highlighted notably as Planktothrix exhibits an overall greater genetic diversity but a smaller genomic plasticity than Microcystis. Our results are shedding light on Planktothrix evolution, phylogeny and physiology in the frame of their diverse life styles.

Frontiers for research on the ecology of plant-pathogenic bacteria: fundamentals for sustainability: Challenges in Bacterial Molecular Plant Pathology.

Methods to ensure the health of crops owe their efficacy to the extent to which we understand the ecology and biology of environmental microorganisms and the conditions under which their interactions with plants lead to losses in crop quality or yield. However, in the pursuit of this knowledge, notions of the ecology of plant-pathogenic microorganisms have been reduced to a plant-centric and agro-centric focus. With increasing global change, i.e. changes that encompass not only climate, but also biodiversity, the geographical distribution of biomes, human demographic and socio-economic adaptations and land use, new plant health problems will emerge via a range of processes influenced by these changes. Hence, knowledge of the ecology of plant pathogens will play an increasingly important role in the anticipation and response to disease emergence. Here, we present our opinion on the major challenges facing the study of the ecology of plant-pathogenic bacteria. We argue that the discovery of markedly novel insights into the ecology of plant-pathogenic bacteria is most likely to happen within a framework of more extensive scales of space, time and biotic interactions than those that currently guide much of the research on these bacteria. This will set a context that is more propitious for the discovery of unsuspected drivers of the survival and diversification of plant-pathogenic bacteria and of the factors most critical for disease emergence, and will set the foundation for new approaches to the sustainable management of plant health. We describe the contextual background of, justification for and specific research questions with regard to the following challenges: Development of terminology to describe plant-bacterial relationships in terms of bacterial fitness. Definition of the full scope of the environments in which plant-pathogenic bacteria reside or survive. Delineation of pertinent phylogenetic contours of plant-pathogenic bacteria and naming of strains independent of their presumed life style. Assessment of how traits of plant-pathogenic bacteria evolve within the overall framework of their life history. Exploration of possible beneficial ecosystem services contributed to by plant-pathogenic bacteria.

The AvrE superfamily: ancestral type III effectors involved in suppression of pathogen-associated molecular pattern-triggered immunity.

The AvrE superfamily of type III effectors (T3Es) is widespread among type III-dependent phytobacteria and plays a crucial role during bacterial pathogenesis. Members of the AvrE superfamily are vertically inherited core effectors, indicating an ancestral acquisition of these effectors in bacterial plant pathogens. AvrE-T3Es contribute significantly to virulence by suppressing pathogen-associated molecular pattern (PAMP)-triggered immunity. They inhibit salicylic acid-mediated plant defences, interfere with vesicular trafficking and promote bacterial growth in planta. AvrE-T3Es elicit cell death in both host and non-host plants independent of any known plant resistance protein, suggesting an original interaction with the plant immune system. Recent studies in yeast have indicated that they activate protein phosphatase 2A and inhibit serine palmitoyl transferase, the first enzyme of the sphingolipid biosynthesis pathway. In this review, we describe the current picture that has emerged from studies of the different members of this fascinating large family.

Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.

Deep sequencing revealed genome-wide single-nucleotide polymorphism and plasmid content of Erwinia amylovora strains isolated in Middle Atlas, Morocco.

Erwinia amylovora causes economic losses that affect pear and apple production in Morocco. Here, we report comparative genomics of four Moroccan E. amylovora strains with the European strain CFBP1430 and North-American strain ATCC49946. Analysis of single nucleotide polymorphisms (SNPs) revealed genetic homogeneity of Moroccan's strains and their proximity to the European strain CFBP1430. Moreover, the collected sequences allowed the assembly of a 65 kpb plasmid, which is highly similar to the plasmid pEI70 harbored by several European E. amylovora isolates. This plasmid was found in 33% of the 40 E. amylovora strains collected from several host plants in 2009 and 2010 in Morocco.

The bacterial effector DspA/E is toxic in Arabidopsis thaliana and is required for multiplication and survival of fire blight pathogen.

The type III effector DspA/E is an essential pathogenicity factor of the phytopathogenic bacterium Erwinia amylovora. We showed that DspA/E was required for transient bacterial growth in nonhost Arabidopsis thaliana leaves, as an E. amylovora dspA/E mutant was unable to grow. We expressed DspA/E in A. thaliana transgenic plants under the control of an oestradiol-inducible promoter, and found that DspA/E expressed in planta restored the growth of a dspA/E mutant. DspA/E expression in these transgenic plants led to the modulation by at least two-fold of the expression of 384 genes, mostly induced (324 genes). Both induced and repressed genes contained high proportions of defence genes. DspA/E expression ultimately resulted in plant cell death without requiring a functional salicylic acid signalling pathway. Analysis of A. thaliana transgenic seedlings expressing a green fluorescent protein (GFP):DspA/E fusion indicated that the fusion protein could only be detected in a few cells per seedling, suggesting the degradation or absence of accumulation of DspA/E in plant cells. Consistently, we found that DspA/E repressed plant protein synthesis when injected by E. amylovora or when expressed in transgenic plants. Thus, we conclude that DspA/E is toxic to A. thaliana: it promotes modifications, among which the repression of protein synthesis could be determinant in the facilitation of necrosis and bacterial growth.

Mutational analysis of a predicted double ß-propeller domain of the DspA/E effector of Erwinia amylovora.

The bacterium Erwinia amylovora causes fire blight, an invasive disease that threatens apple trees, pear trees and other plants of the Rosaceae family. Erwinia amylovora pathogenicity relies on a type III secretion system and on a single effector DspA/E. This effector belongs to the widespread AvrE family of effectors whose biological function is unknown. In this manuscript, we performed a bioinformatic analysis of DspA/E- and AvrE-related effectors. Motif search identified nuclear localization signals, peroxisome targeting signals, endoplasmic reticulum membrane retention signals and leucine zipper motifs, but none of these motifs were present in all the AvrE-related effectors analysed. Protein threading analysis, however, predicted a conserved double ß-propeller domain in the N-terminal part of all the analysed effector sequences. We then performed a random pentapeptide mutagenesis of DspA/E, which led to the characterization of 13 new altered proteins with a five amino acids insertion. Eight harboured the insertion inside the predicted ß-propeller domain and six of these eight insertions impaired DspA/E stability or function. Conversely, the two remaining insertions generated proteins that were functional and abundantly secreted in the supernatant suggesting that these two insertions stabilized the protein.

Fire blight: applied genomic insights of the pathogen and host.

The enterobacterial phytopathogen Erwinia amylovora causes fire blight, an invasive disease that threatens a wide range of commercial and ornamental Rosaceae host plants. The response elicited by E. amylovora in its host during disease development is similar to the hypersensitive reaction that typically leads to resistance in an incompatible host-pathogen interaction, yet no gene-for-gene resistance has been described for this host-pathogen system. Comparative genomic analysis has found an unprecedented degree of genetic uniformity among strains of E. amylovora, suggesting that the pathogen has undergone a recent genetic bottleneck. The genome of apple, an important host of E. amylovora, has been sequenced, creating new opportunities for the study of interactions between host and pathogen during fire blight development and for the identification of resistance genes. This review includes recent advances in the genomics of both host and pathogen.

EDS1 contributes to nonhost resistance of Arabidopsis thaliana against Erwinia amylovora.

Erwinia amylovora causes fire blight in rosaceous plants. In nonhost Arabidopsis thaliana, E. amylovora triggers necrotic symptoms associated with transient bacterial multiplication, suggesting either that A. thaliana lacks a susceptibility factor or that it actively restricts E. amylovora growth. Inhibiting plant protein synthesis at the time of infection led to an increase in necrosis and bacterial multiplication and reduced callose deposition, indicating that A. thaliana requires active protein synthesis to restrict E. amylovora growth. Analysis of the callose synthase-deficient pmr4-1 mutant indicated that lack of callose deposition alone did not lead to increased sensitivity to E. amylovora. Transcriptome analysis revealed that approximately 20% of the genes induced following E. amylovora infection are related to defense and signaling. Analysis of mutants affected in NDR1 and EDS1, two main components of the defense-gene activation observed, revealed that E. amylovora multiplied ten times more in the eds1-2 mutant than in the wild type but not in the ndr1-1 mutant. Analysis of mutants affected in three WRKY transcription factors showing EDS1-dependent activation identified WRKY46 and WRKY54 as positive regulators and WRKY70 as a negative regulator of defense against E. amylovora. Altogether, we show that EDS1 is a positive regulator of nonhost resistance against E. amylovora in A. thaliana and hypothesize that it controls the production of several effective defenses against E. amylovora through the action of WRKY46 and WRKY54, while WRKY70 acts as a negative regulator.

The HrpN effector of Erwinia amylovora, which is involved in type III translocation, contributes directly or indirectly to callose elicitation on apple leaves.

Erwinia amylovora is responsible for fire blight of apple and pear trees. Its pathogenicity depends on a type III secretion system (T3SS) mediating the translocation of effectors into the plant cell. The DspA/E effector suppresses callose deposition on apple leaves. We found that E. amylovora and Pseudomonas syringae DC3000 tts mutants or peptide flg22 do not trigger callose deposition as strongly as the dspA/E mutant on apple leaves. This suggests that, on apple leaves, callose deposition is poorly elicited by pathogen-associated molecular patterns (PAMPs) such as flg22 or other PAMPs harbored by tts mutants and is mainly elicited by injected effectors or by the T3SS itself. Callose elicitation partly depends on HrpW because an hrpW-dspA/E mutant elicits lower callose deposition than a dspA/E mutant. Furthermore, an hrpN-dspA/E mutant does not trigger callose deposition, indicating that HrpN is required to trigger this plant defense reaction. We showed that HrpN plays a general role in the translocation process. Thus, the HrpN requirement for callose deposition may be explained by its role in translocation: HrpN could be involved in the translocation of other effectors inducing callose deposition. Furthermore, HrpN may also directly contribute to the elicitation process because we showed that purified HrpN induces callose deposition.