Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 x 10(6) conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens.

Plant responses to enemies are coordinated by several interacting signaling systems. Molecular and genetic studies with mutants and exogenous signal application suggest that jasmonate (JA)-, salicylate (SA)-, and ethylene (ET)-mediated pathways modulate expression of portions of the defense phenotype in Arabidopsis (Arabidopsis thaliana), but have not yet linked these observations directly with plant responses to insect attack. We compared the glucosinolate (GS) profiles of rosette leaves of 4-week-old mutant and transgenic Arabidopsis (Columbia) plants compromised in these three major signaling pathways, and characterized responses by those plants to feeding by two phloem-feeding aphids (generalist Myzus persicae and specialist Brevicoryne brassicae) and one generalist caterpillar species (Spodoptera exigua Hubner). Blocked JA signaling in coronatine-insensitive (coi1) and enhanced expression of SA-signaled disease resistance in hypersensitive response-like (hrl1) mutants reduced constitutive GS concentrations, while blocking SA signaling at the mediator protein npr1 mutant (NPR) increased them. There was no significant impact on constitutive GS contents of blocking ET signaling (at ETresistant [etr1]) or reducing SA concentrations (nahG transgene). We found increased GS accumulation in response to insect feeding, which required functional NPR1 and ETR1 but not COI1 or SA. Insect feeding caused increases primarily in short-chain aliphatic methylsulfinyl GS. By contrast, responses to exogenous JA, a frequent experimental surrogate for insect attack, were characterized by an increase in indolyl GS. Insect performance, measured as population increase or weight increase, was negatively related to GS levels, but we found evidence that other, ET-regulated factors may also be influential. Plant resistance to (consumption by) S. exigua was not related to insect growth because some plant chemistries inhibited growth while others inhibited feeding. These major signaling pathways modulate Arabidopsis GS accumulation and response to both phloem-feeding and chewing insects, often antagonistically; NPR appears to be central to these interactions. Our results indicate that exogenous signal application and plant consumption measures may not provide useful measures of plant responses to actual insect feeding.Plants have developed diverse defense mechanisms for dealing with enemies. Like all plants in the Brassicaceae, Arabidopsis (Arabidopsis thaliana) produces secondary metabolites, including glucosinolates (GS), phenolics, and terpenoids (Halkier, 1999;Madhuri and Reddy, 1999;Harrewijn et al., 2001;Mikkelsen et al., 2003), which may have defensive functions. GS are found mainly in the order Brassicales, which includes the Brassicaceae (Giamoustaris and Mithen, 1995;Tierens et al., 2001), where they function in defense against herbivores and pathogens. GS are sulfonated thioglycosides comprising a common glycone moiety with a variable aglycone side chain. Three major classes of GS are distinguished: aliphatic GS derived princi...

SummaryResistance to Turnip Crinkle Virus (TCV) in Arabidopsis ecotype Dijon (Di)-17 is conferred by the resistance gene HRT and a recessive locus rrt. In Di-17, TCV elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes and high levels of salicylic acid (SA). We have previously shown that HRT-mediated resistance to TCV is dependent on SA-mediated signal transduction and that increased levels of SA confer enhanced resistance to TCV via upregulation of the HRT gene. Here we show that HRT-mediated HR and resistance are dependent on light. A dark treatment immediately following TCV inoculation suppressed HR, resistance and activation of the majority of the TCV-induced genes. However, the absence of light did not affect either TCV-induced elevated levels of free SA or the expression of HRT. Interestingly, in the dark, transgenic plants overexpressing HRT showed susceptibility, but overexpression of HRT coupled with high levels of endogenous SA resulted in pronounced resistance. Consistent with these results is the finding that exogenous application of SA prior to TCV inoculation partially overcame the requirement for light. Light was also required for N gene-mediated HR and resistance to Tobacco Mosaic Virus, suggesting that it is an important factor which may be generally required during defense signaling.

SummaryIn Arabidopsis, the GH3-like gene family consists of 19 members, several of which have been shown to adenylate the plant hormones jasmonic acid, indole acetic acid and salicylic acid (SA). In some cases, this adenylation has been shown to catalyze hormone conjugation to amino acids. Here we report molecular characterization of the GH3-LIKE DEFENSE GENE 1 (GDG1), a member of the GH3-like gene family, and show that GDG1 is an important component of SA-mediated defense against the bacterial pathogen Pseudomonas syringae. Expression of GDG1 is induced earlier and to a higher level in response to avirulent pathogens compared to virulent pathogens. gdg1 null mutants are compromised in several pathogen defense responses, including activation of defense genes and resistance against virulent and avirulent bacterial pathogens. Accumulation of free and glucoside-conjugated SA (SAG) in response to pathogen infection is compromised in gdg1 mutants. All defense-related phenotypes of gdg1 can be rescued by external application of SA, suggesting that gdg1 mutants are defective in the SA-mediated defense pathway(s) and that GDG1 functions upstream of SA. Our results suggest that GDG1 contributes to both basal and resistance gene-mediated inducible defenses against P. syringae (and possibly other pathogens) by playing a critical role in regulating the levels of pathogen-inducible SA. GDG1 is allelic to the PBS3 (avrPphB susceptible) gene.

SummaryProtein kinases play important roles in relaying information from perception of a signal to the effector genes in all organisms. Cysteine-rich receptor-like kinases (CRKs) constitute a sub-family of plant receptor-like kinases (RLKs) with more than 40 members that contain the novel C-X8-C-X2-C motif (DUF26) in the extracellular domains. Here we report molecular characterization of one member of this gene family, CRK13. Expression of this gene is induced more quickly and strongly in response to the avirulent compared with the virulent strains of Pseudomonas syringae, and peaks within 4 h after pathogen infection. In response to dexamethasone (DEX) treatment, plants expressing the CRK13 gene from a DEX-inducible promoter exhibited all tested features of pathogen defense activation, including rapid tissue collapse, accumulation of high levels of several defenserelated gene transcripts including PR1, PR5 and ICS1, and accumulation of salicylic acid (SA). In addition, these plants suppressed growth of virulent pathogens by about 20-fold compared with the wild-type Col-0. CRK13-conferred pathogen resistance is salicylic acid-dependent. Gene expression analysis using custom cDNA microarrays revealed a remarkable overlap between the expression profiles of the plants overexpressing CRK13 and the plants treated with Pst DC3000 (avrRpm1). Our studies suggest that upregulation of CRK13 leads to hypersensitive response-associated cell death, and induces defense against pathogens by causing increased accumulation of salicylic acid.

SummaryDefence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA-and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SAdepleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel-blot analysis of RNA isolated separately from the lesion + and the lesion ± leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.

Pathogen-induced plant responses include changes in both volatile and non-volatile secondary metabolites. To characterize the role of bacterial pathogenesis in plant volatile emissions, tobacco plants, Nicotiana tabacum L. K326, were inoculated with virulent, avirulent, and mutant strains of Pseudomonas syringae. Volatile compounds released by pathogen-inoculated tobacco plants were collected, identified, and quantified. Tobacco plants infected with the avirulent strains P. syringae pv. maculicola ES4326 (Psm ES4326) or pv. tomato DC3000 (Pst DC3000), emitted quantitatively different, but qualitatively similar volatile blends of (E)-beta-ocimene, linalool, methyl salicylate (MeSA), indole, caryophyllene, beta-elemene, alpha-farnesene, and two unidentified sesquiterpenes. Plants treated with the hrcC mutant of Pst DC3000 (hrcC, deficient in the type-III secretion system) released low levels of many of the same volatile compounds as in Psm ES4326- or Pst DC3000-infected plants, with the exception of MeSA, which occurred only in trace amounts. Interaction of the virulent pathogen P. syringae pv. tabaci (Pstb), with tobacco plants resulted in a different volatile blend, consisting of MeSA and two unidentified sesquiterpenes. Overall, maximum volatile emissions occurred within 36 h post-inoculation in all the treatments except for the Pstb infection that produced peak volatile emissions about 60 h post-inoculation. (E)-beta-Ocimene was released in a diurnal pattern with the greatest emissions during the day and reduced emissions at night. Both avirulent strains, Psm ES4326 and Pst DC3000, induced accumulation of free salicylic acid (SA) within 6 h after inoculation and conjugated SA within 60 h and 36 h respectively. In contrast, SA inductions by the virulent strain Pstb occurred much later and conjugated SA increased slowly for a longer period of time, while the hrcC mutant strain did not trigger free and conjugated SA accumulations in amounts significantly different from control plants. Jasmonic acid, known to induce plant volatile emissions, was not produced in significantly higher levels in inoculated plants compared to the control plants in any treatments, indicating that induced volatile emissions from tobacco plants in response to P. syringae are not linked to changes in jasmonic acid.

Abstract. There is little comprehensive information on the distribution of root systems among coexisting species, despite the expected importance of those distributions in determining the composition and diversity of plant communities. This gap in knowledge is particularly acute for grasslands, which possess large numbers of species with morphologically indistinguishable roots. In this study we adapted a molecular method, fluorescent fragment length polymorphism, to identify root fragments and determine species root distributions in two grasslands in Yellowstone National Park (YNP). Aboveground biomass was measured, and soil cores (2 cm in diameter) were collected to depths of 40 cm and 90 cm in an upland, dry grassland and a mesic, slope-bottom grassland, respectively, at peak foliar expansion. Cores were subdivided, and species that occurred in each 10-cm interval were identified. The results indicated that the average number of species in 10-cm intervals (31 cm 3 ) throughout the sampled soil profile was 3.9 and 2.8 species at a dry grassland and a mesic grassland, respectively. By contrast, there was an average of 6.7 and 14.1 species per 0.5 m 2 , determined by the presence of shoot material, at dry and mesic sites, respectively. There was no relationship between soil depth and number of species per 10-cm interval in either grassland, despite the exponential decline of root biomass with soil depth at both sites. There also was no relationship between root frequency (i.e., the percentage of samples in which a species occurred) and soil depth for the vast majority of species at both sites. The preponderance of species were distributed throughout the soil profile at both sites. Assembly analyses indicated that species root occurrences were randomly assorted in all soil intervals at both sites, with the exception that Festuca idahoensis segregated from Artemisia tridentata and Pseudoroegnaria spicata in 10-20 cm soil at the dry grassland. Root frequency throughout the entire sampled soil profile was positively associated with shoot biomass among species. Together these results indicated the importance of large, well-proliferated root systems in establishing aboveground dominance. The findings suggest that spatial belowground segregation of species probably plays a minor role in fostering resource partitioning and species coexistence in these YNP grasslands.