John W. Mansfield

Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease

The avrPphF gene was cloned from Pseudomonas syringae pathovar phaseolicola (Pph) races 5 and 7, based on its ability to confer avirulence towards bean cultivars carrying the R1 gene for halo‐blight resistance, such as Red Mexican. avrPphF comprised two open reading frames, which were both required for function, and was located on a 154 kb plasmid (pAV511) in Pph. Strain RW60 of Pph, lacking pAV511, displayed a loss in virulence to a range of previously susceptible cultivars such as Tendergreen and Canadian Wonder. In Tendergreen virulence was restored to RW60 by avrPphF alone, whereas subcloned avrPphF in the absence of pAV511 greatly accelerated the hypersensitive resistance reaction caused by RW60 in Canadian Wonder. A second gene from pAV511, avrPphC, which controls avirulence to soybean, was found to block the activity of avrPphF in Canadian Wonder, but not in Red Mexican. avrPphF also conferred virulence in soybean. The multiple functions of avrPphF illustrate how effector proteins from plant pathogens have evolved to be recognized by R gene products and, therefore, be classified as encoded by avirulence genes.

Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H2O2-and o-dianisidine/H2O2-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.

Early events in host-pathogen interactions

Research focused on early events in host-pathogen interactions has provided new insights into fundamental aspects of microbial pathogenicity and plant responses. Considerable progress has been made in understanding regulation of the delivery of pathogenicity determinants from bacteria into plant cells, signal cascades involved in fungal pathogenicity, the co-ordinating role of the plant cytoskeleton in plant defence and calcium flux as a primary signalling function during the hypersensitive reaction.

The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ

The type III secretion system (TTSS) is an essential requirement for the virulence of many Gram‐negative bacteria infecting plants, animals and man. Pathogens use the TTSS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cell, where the effectors subvert host defences. Plant pathogens have to translocate their effector proteins through the plant cell wall barrier. The best candidates for directing effector protein traffic are bacterial appendages attached to the membrane‐bound components of the TTSS. We have investigated the protein secretion route in relation to the TTSS appendage, termed the Hrp pilus, of the plant pathogen Pseudomonas syringae pv. tomato. By pulse expression of proteins combined with immunoelectron microscopy, we show that the Hrp pilus elongates by the addition of HrpA pilin subunits at the distal end, and that the effector protein HrpZ is secreted only from the pilus tip. Our results indicate that both HrpA and HrpZ travel through the Hrp pilus, which functions as a conduit for the long‐distance translocation of effector proteins.

Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and a gain in cultivar-specific virulence.

The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola (Psph), is differentiated into nine races based on the presence or absence of five avirulence (avru200a) genes in the bacterium, which interact with corresponding resistance genes, R1–R5, in Phaseolus vulgaris. The resistance gene R2 is matched by avrPphE, which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph. Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph. DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104u2003bp in the allele in race 8. We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE. Agrobacterium tumefaciens‐directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene‐specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta, whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plants response. Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum. The avirulence gene avrPphB, which matches the R3 resistance gene, also caused a gene‐specific HR following expression in the plant after delivery by A. tumefaciens.

Avirulence genes from Pseudomonas syringae pathovars phaseolicola and pisi confer specificity towards both host and non-host species☆

Abstract Avirulence genes from Pseudomonas syringae pathovars phaseolicola and pisi which determine specificity towards cultivars of their respective host species (bean and pea), were found also to determine specificity towards the non-host species. Transconjugants of Ps.s. pv. pisi harbouring clones containing avrPph 3, previously isolated from race 3 of Ps.s. pv. phaseolicola , induced a hypersensitive reaction (HR) in all pea cultivars tested. Clones containing avrPpi 2, previously isolated from Ps.s. pv. pisi race 2, caused Ps.s. pv. phaseolicola to induce a rapid HR in most cultivars of bean but in cv. Seafarer a markedly slower incompatible response was observed. Transconjugants of the heterologous pathovar harbouring avrPpi 2 or avrPph 3, therefore, induced resistance patterns in non-host cultivars which were unlike those of any known race of the homologous pathogen. The quantitatively different resistance responses of some bean cultivars were further characterized by analyses of phytoalexin accumulation and bacterial multiplication in pod tissue. Responses of F 2 progeny of crosses between bean cultivars towards transconjugants harbouring avrPpi 2 suggested the presence of a dominant allele at a single locus regulating the rapid HR characteristic of resistance in cv. Canadian Wonder and a dominant allele at an independent second locus regulating the slower resistance reaction of cv. Seafarer. It appears that a one gene for two genes relationship is involved in the avrPpi 2-bean interaction. Clones carrying two other avirulence genes, avrPph 2 and avrPpi 3, from Ps.s. pv. phaseolicola and Ps.s. pv. pisi , respectively, had no effect on the virulence of heterologous pathovars towards any of the cultivars tested.

Histological, physiological and genetical studies of the responses of leaves and pods of Phaseolus vulgaris to three races of Pseudomonas syringae pv. phaseolicola and to Pseudomonas syringae pv. coronafaciens

Abstract The responses of bean leaves and pods to races 1, 2 and 3 of Pseudomonas syringae pv. phaseolicola and to P.s. pv. coronafaciens were compared. Compatible and incompatible reactions were more clearly distinguished in pods than in leaves. Ultrastructural studies showed that a prolonged biotrophic phase of infection occurred in susceptible pods. Colonies of avirulent and virulent bacteria were surrounded by acidic polysaccharides in the intercellular spaces, A rapid hypersensitive reaction (HR) occurred in tissues of Phaseolus vulgaris cv. Tendergreen inoculated with race 3 or P.s. pv. coronafaciens . The intensity of the reaction was indicated by the low numbers of bacteria required for symptom development, shorter induction times than for race 1 in P. vulgaris cv. Red Mexican and higher concentrations of phaseollin recovered from cv. Tendergreen than other cultivars Experiments with mixed inocula suggested that production of elicitors of the HR rather than suppression of the resistant reaction by virulent isolates occurred. No evidence was obtained for the release of diffusible elicitors of the HR from avirulent bacteria or from responding tissue. Mutants of race 3 with altered virulence were recovered following treatment with nitrosoguanidine causing c . 7% of survivors to be auxotrophs. Three categories of mutants were recovered after screening on pods of cultivars Canadian Wonder, Red Mexican and Tendergreen (1) mutants with altered varietal specificity, they had lost the ability to cause the HR in cv. Tendergreen but caused large watersoaked lesions in all cultivars tested, (2) forms which caused small watersoaked lesions in the susceptible cultivars, Canadian Wonder and Red Mexican, but caused the HR in cv. Tendergreen, and (3) non-pathogenic forms failing to cause symptoms in any cultivar.

Excision from tRNA genes of a large chromosomal region, carrying avrPphB, associated with race change in the bean pathogen, Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola (Pph) race 4 strain 1302A carries avirulence gene avrPphB. Strain RJ3, a sectoral variant from a 1302A culture, exhibited an extended host range in cultivars of bean and soybean resulting from the absence of avrPphB from the RJ3 chromosome. Complementation of RJ3 with avrPphB restored the race 4 phenotype. Both strains showed similar in planta growth in susceptible bean cultivars. Analysis of RJ3 indicated loss of >u200a40u2003kb of DNA surrounding avrPphB. Collinearity of the two genomes was determined for the left and right junctions of the deleted avrPphB region; the left junction is ≈u200a19u2003kb and the right junction >u200a20u2003kb from avrPphB in 1302A. Sequencing revealed that the region containing avrPphB was inserted into a tRNALYS gene, which was re‐formed at the right junction in strain 1302A. A putative lysine tRNA pseudogene (ΨtRNALYS) was found at the left junction of the insertion. All tRNA genes were in identical orientation in the chromosome. Genes near the left junction exhibited predicted protein homologies with gene products associated with a virulence locus of the periodontal pathogen Actinobacillus actinomycetemcomitans. Specific oligonucleotide primers that differentiate 1302A from RJ3 were designed and used to demonstrate that avrPphB was located in different regions of the chromosome in other strains of Pph. Deletion of a large region of the chromosome containing an avirulence gene represents a new route to race change in Pph.

The phytoalexin response of lettuce to challenge by Botrytis cinerea, Bremia lactucae and Pseudomonas syringae pv. phaseolicola

Lettucenin A was confirmed to be the principal phytoalexin in lettuce. The accumulation of lettucenin A was directly related to the number of cells undergoing the hypersensitive reaction (HR) during incompatible interactions with Bremia lactucae governed by the Dm5/8 and Dm7 resistance genes. Very little phytoalexin accumulation occurred during the successful colonization of cotyledons by a virulent isolate of B. lactucae. The HR to Pseudomonas syringae pathovar phaseolicola was also associated with the localized accumulation of lettucenin A. Inoculation of leaves with conidia of Botrytis cinerea caused the formation of limited lesions within which fungal growth was restricted. Lettucenin A reached high concentrations in tissue bearing limited lesions and within inoculum droplets on the leaf surface. Mycelial inocula of B. cinerea produced spreading lesions. During successful colonization by B. cinerea an initial increase in lettucenin A concentration at infection sites was followed by a decrease as tissue became completely colonized and rotted by the grey mould fungus. The success or failure of B. cinerea to invade lettuce appeared to be based on a balance between phytoalexin accumulation and degradation. Lettucenin A at 10−6 m concentrations was found to possess considerable activity against B. lactucae, B. cinerea and P. syringae pv. phaseolicola and probably has an important role in the resistance of lettuce tissues to microbial colonization.

NopB, a Type III Secreted Protein of Rhizobium sp. Strain NGR234, Is Associated with Pilus-Like Surface Appendages

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.