Kim Findlay

Biofilm dispersal in Xanthomonas campestris is controlled by cell–cell signaling and is required for full virulence to plants

The rpf gene cluster of Xanthomonas campestris pathovar campestris (Xcc) is required for the pathogenesis of this bacterium to plants. Several rpf genes are involved in the coordinate positive regulation of the production of virulence factors mediated by the small diffusible molecule DSF (for diffusible signal factor). RpfF directs the synthesis of DSF, and a two-component sensory transduction system comprising RpfC and RpfG has been implicated in the perception of the DSF signal and signal transduction. In L medium, rpfF, rpfG, rpfC, and rpfGHC mutants grew as matrix-enclosed aggregates, whereas the wild type grew in a dispersed planktonic fashion. Synthesis of the extracellular polysaccharide xanthan was required for aggregate formation. Addition of DSF triggered dispersion of the aggregates formed by the rpfF strain, but not those of rpf strains defective in DSF signal transduction. An extracellular enzyme from Xcc whose synthesis was positively controlled by the DSF/rpf system could disperse the aggregates produced by all rpf strains. The enzyme was identified as the single endo-β-1,4-mannanase encoded by the Xcc genome. This enzyme had no detectable activity against soluble xanthan. The endo-β-1,4-mannanase was required for the full virulence of Xcc to plants. On the basis of this model system, we propose that one role of the β-mannanase during disease is to promote transitions from an aggregated or biofilm lifestyle to a planktonic lifestyle in response to the DSF signal.

The Accumulation of Oleosins Determines the Size of Seed Oilbodies in Arabidopsis

We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.

Virus-Induced Silencing of a Plant Cellulose Synthase Gene

Specific cDNA fragments corresponding to putative cellulose synthase genes (CesA) were inserted into potato virus X vectors for functional analysis in Nicotiana benthamiana by using virus-induced gene silencing. Plants infected with one group of cDNAs had much shorter internode lengths, small leaves, and a “dwarf” phenotype. Consistent with a loss of cell wall cellulose, abnormally large and in many cases spherical cells ballooned from the undersurfaces of leaves, particularly in regions adjacent to vascular tissues. Linkage analyses of wall polysaccharides prepared from infected leaves revealed a 25% decrease in cellulose content. Transcript levels for at least one member of the CesA cellulose synthase gene family were lower in infected plants. The decrease in cellulose content in cell walls was offset by an increase in homogalacturonan, in which the degree of esterification of carboxyl groups decreased from ∼50 to ∼33%. The results suggest that feedback loops interconnect the cellular machinery controlling cellulose and pectin biosynthesis. On the basis of the phenotypic features of the infected plants, changes in wall composition, and the reduced abundance of CesA mRNA, we concluded that the cDNA fragments silenced one or more cellulose synthase genes.

Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

Tensile Properties of Arabidopsis Cell Walls Depend on Both a Xyloglucan Cross-Linked Microfibrillar Network and Rhamnogalacturonan II-Borate Complexes

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.

An Arabidopsis GPI-Anchor Plasmodesmal Neck Protein with Callose Binding Activity and Potential to Regulate Cell-to-Cell Trafficking

Plasmodesmata (Pds) traverse the cell wall to establish a symplastic continuum through most of the plant. Rapid and reversible deposition of callose in the cell wall surrounding the Pd apertures is proposed to provide a regulatory process through physical constriction of the symplastic channel. We identified members within a larger family of X8 domain–containing proteins that targeted to Pds. This subgroup of proteins contains signal sequences for a glycosylphosphatidylinositol linkage to the extracellular face of the plasma membrane. We focused our attention on three closely related members of this family, two of which specifically bind to 1,3-β-glucans (callose) in vitro. We named this family of proteins Pd callose binding proteins (PDCBs). Yellow fluorescent protein-PDCB1 was found to localize to the neck region of Pds with potential to provide a structural anchor between the plasma membrane component of Pds and the cell wall. PDCB1, PDCB2, and PDCB3 had overlapping and widespread patterns of expression, but neither single nor combined insertional mutants for PDCB2 and PDCB3 showed any visible phenotype. However, increased expression of PDCB1 led to an increase in callose accumulation and a reduction of green fluorescent protein (GFP) movement in a GFP diffusion assay, identifying a potential association between PDCB-mediated callose deposition and plant cell-to-cell communication.

Escherichia coli Strains Blocked in Tat-Dependent Protein Export Exhibit Pleiotropic Defects in the Cell Envelope

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates.

The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1

In contrast to many mammalian pathogens, potential bacterial pathogens of plants remain outside the host cell. The plant must, therefore, promote an active resistance mechanism to combat the extracellular infection. How this resistance against bacteria is manifested and whether similar processes mediate basal, gene-for-gene, and salicylate-associated defense, however, are poorly understood. Here, we identify a specific plasma membrane syntaxin, NbSYP132, as a component contributing to gene-for-gene resistance in Nicotiana benthamiana. Silencing NbSYP132 but not NbSYP121, the apparent orthologue of a syntaxin required for resistance to powdery mildew fungus, compromised AvrPto-Pto resistance. Because syntaxins may play a role in secretion of proteins to the extracellular space, we performed a limited proteomic analysis of the apoplastic fluid. We found that NbSYP132-silenced plants were impaired in the accumulation of at least a subset of pathogenesis-related (PR) proteins in the cell wall. These results were confirmed by both immunoblot analysis and imunolocalization of a PR protein, PR1a. These results implicate NbSYP132 as the cognate target soluble N-ethylmaleimide-sensitive factor attachment protein receptor for exocytosis of vesicles containing antimicrobial PR proteins. NbSYP132 also contributes to basal and salicylate-associated defense, indicating that SYP132-dependent secretion is a component of multiple forms of defense against bacterial pathogens in plants.

A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp.

A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well‐conserved region 2.2 of the eubacterial σ70 family. Homologues were present in other Streptomyces spp., and that of the genetically well‐studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to σB of Bacillus subtilis. However, while σB is involved in stationary‐phase gene expression and in the general stress response in B. subtilis, σF affects morphological differentiation in Streptomyces, Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. σF is proposed to control the late stages of spore development in Streptomyces.

The Receptor-Like Kinase SERK3/BAK1 Is Required for Basal Resistance against the Late Blight Pathogen Phytophthora infestans in Nicotiana benthamiana

Background The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. Principal Findings We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. Conclusions/Significance We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.