A developmental response to pathogen infection in Arabidopsis

We present evidence that susceptible Arabidopsis plants accelerate their reproductive development and alter their shoot architecture in response to three different pathogen species. We infected 2-week-old Arabidopsis seedlings with two bacterial pathogens, Pseudomonas syringae and Xanthomonas campestris, and an oomycete, Peronospora parasitica. Infection with each of the three pathogens reduced time to flowering and the number of aerial branches on the primary inflorescence. In the absence of competition, P. syringae and P. parasitica infection also increased basal branch development. Flowering time and branch responses were affected by the amount of pathogen present. Large amounts of pathogen caused the most dramatic changes in the number of branches on the primary inflorescence, but small amounts of P. syringae caused the fastest flowering and the production of the most basal branches. RPS2 resistance prevented large changes in development when it prevented visible disease symptoms but not at high pathogen doses and when substantial visible hypersensitive response occurred. These experiments indicate that phylogenetically disparate pathogens cause similar changes in the development of susceptible Arabidopsis. We propose that these changes in flowering time and branch architecture constitute a general developmental response to pathogen infection that may affect tolerance of and/or resistance to disease.     

A plant gene up-regulated at rust infection sites

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a beta-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%-82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a delta1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.     

Genome-wide association mapping in Arabidopsis identifies previously known flowering time and pathogen resistance genes

There is currently tremendous interest in the possibility of using genome-wide association mapping to identify genes responsible for natural variation, particularly for human disease susceptibility. The model plant Arabidopsis thaliana is in many ways an ideal candidate for such studies, because it is a highly selfing hermaphrodite. As a result, the species largely exists as a collection of naturally occurring inbred lines, or accessions, which can be genotyped once and phenotyped repeatedly. Furthermore, linkage disequilibrium in such a species will be much more extensive than in a comparable outcrossing species. We tested the feasibility of genome-wide association mapping in A. thaliana by searching for associations with flowering time and pathogen resistance in a sample of 95 accessions for which genome-wide polymorphism data were available. In spite of an extremely high rate of false positives due to population structure, we were able to identify known major genes for all phenotypes tested, thus demonstrating the potential of genome-wide association mapping in A. thaliana and other species with similar patterns of variation. The rate of false positives differed strongly between traits, with more clinal traits showing the highest rate. However, the false positive rates were always substantial regardless of the trait, highlighting the necessity of an appropriate genomic control in association studies.     

Identification of Arabidopsis subtilisin-like serine protease specifically expressed in root stele by gene trapping

Xylem plays a role not only in the transport of water and nutrients but also in the regulation of growth and development through the transport of biologically active substances. In addition to mineral salts, xylem sap contains hormones, organic nutrients and proteins. However, the physiological functions of most of those substances remain unclear. To explore genes involved in xylem sap production, we identified Arabidopsis genes expressed in the root stele of the root hair zone from gene-trap lines by randomly inserting the β-glucuronidase gene into the genome. Among 26 000 gene-trap lines, we found that 10 lines had β-glucuronidase (GUS) staining predominantly in the root stele of the root hair zone and no GUS staining in the shoots. Of these 10 lines, 2 lines showed that gene-trap tags inserted into the promoter region of the same gene, denoted Arabidopsis thaliana subtilase 4.12( AtSBT4.12 ). Analysis of AtSBT4.12 promoter using an pAtSBT4.12 ::β-glucuronidase transgenic line showed that the AtSBT4.12 gene was expressed only in the root stele of the root hair zone. AtSBT4.12 expression in roots was increased by application of methyl jasmonate. Subtilase proteins are commonly detected in proteomic analyses of xylem sap from various plant species, including Brassica napus , a relative of Arabidopsis . These results suggest that AtSBT4.12 may be a protein localized in the apoplast of root stele including xylem vessel and involved in stress responses in Arabidopsis roots.     

Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection

The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC(50) = 1-20 microM) against fungal and bacterial plant pathogens. It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria. The corresponding StSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point = 9.16) and 66 amino acid residues long (molecular weight of 7,025). The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment. Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and Erwinia chrysanthemi. These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers.     

Cloning of an Arabidopsis patatin-like gene, STURDY, by activation T-DNA tagging

Activation T-DNA tagging can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene. We identified an activation-tagged mutant, sturdy, exhibiting a stiff inflorescence stem, thicker leaves, shorter siliques, larger seeds, round-shaped flowers, and delayed growth. It is most important that unlike its wild-type counterpart, this mutant is less prone to lodging. Cloning of STURDY revealed that in sturdy, there is an open reading frame containing a single intron encoding a patatin-like homolog. The T-DNA is inserted into the 3' region of the second exon. The mutant phenotype was shown to be the result of overexpression of STURDY by mRNA analysis and transgenic studies. Preliminary histological studies have revealed an increase in cell number in the inflorescence stem of mutant plants; however, additional studies are needed to better understand the overexpression phenotype.     

Complex regulation of gene expression, photosynthesis and sugar levels by pathogen infection in tomato

The infection of plants with pathogens results in the induction of defence reactions as well as changes in carbohydrate metabolism. On the one hand, the pathogen attempts to manipulate the carbohydrate metabolism of the plant for its own advantage. On the other, the plant has to reorganize carbon fluxes to ensure fight against the pathogen. In order to further investigate the connection between pathogen infection and carbohydrate metabolism, the effects of two types of pathogen, biotrophic and necrotrophic, on gene expression, endogenous sugar levels and photosynthesis of tomato plants were analysed. Photosynthetic gene expression was downregulated on infection with Pseudomonas syringae and Botrytis cinerea . In contrast, expression of a sink-specific gene encoding a cell wall invertase and of defence genes was induced by both pathogens. These results provide evidence for a co-regulation of defence, sink and photosynthetic gene expression in planta in response to both types of pathogen. The brassinosteroid-containing plant restorative ComCat enhanced resistance against B. cinerea and counter-regulated the repression of photosynthetic gene expression. Endogenous sugar levels decreased and the hexose to sucrose ratio increased on treatment with B. cinerea . The application of chlorophyll fluorescence imaging revealed the spatio-temporal heterogeneity of the pathogen response. At 24 h after infection, inhibition of photosynthetic electron transport was restricted to the direct vicinity of the infection site, which was surrounded by a circle of increased photosynthetic activity. The photosynthesis of the remaining leaf was not affected at this stage. These results show the usefulness of chlorophyll fluorescence imaging for the assessment of the complex spatio-temporal changes and for the definition of the areas relevant for other types of determination, e.g. gene expression.     

Intermediate trapping on a mutant retaining alpha-galactosyltransferase identifies an unexpected aspartate residue

Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate.     

The effects of pathogen infection and mutation on life-history characters in Arabidopsis thaliana

The nature of the interaction among deleterious mutations is important to models in many areas of evolutionary biology. In addition, interactions between genetic and environmental factors may affect the predictions of such models. Individuals of unknown genotypes of Arabidopsis thaliana, ecotype Marburg, were exposed to five levels of chemical (EMS) mutagenesis and three levels of Pseudomonas syringae infection. Survival, growth and flowering characteristics of each individual were measured. The logarithm of fitness is expected to be a linear function of mutation number if mutations act independently. Furthermore, the expected number of mutations should be approximately a linear function of time of exposure to mutagen. Therefore, nonlinear effects of mutagen exposure on the logarithm of fitness characters would suggest epistasis between mutations. Similarly, if pathogen infection and mutation act independently of each other, their effects should be additive on a log scale. Statistical interactions between these factors would suggest they do not act independently; particularly, if highly mutated individuals suffer more when infected than do less mutated individuals, this suggests that pathogens and mutations act synergistically. Pseudomonas-infected individuals were shown to have an increased probability of flowering under conditions of short day length, but to ultimately produce fewer flowers than uninfected individuals. This suggests a plastic response to stress and, despite that response, an ultimately deleterious effect of infection on fitness. Leaf rosette growth was negatively and linearly related to the expected number of mutations, and the effects of mutation on different life-cycle stages appeared to be uncorrelated. No significant interactions between pathogen and mutation main effects were found. These results suggest that mutations act multiplicatively with each other and with pathogen infection in determining individual fitness.