The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato.

It has been proposed that cloned plant disease resistance genes could be transferred from resistant to susceptible plant species to control important crop plant diseases. The recently cloned N gene of tobacco confers resistance to the viral pathogen, tobacco mosaic virus. We generated transgenic tomato plants bearing the N gene and demonstrate that N confers a hypersensitive response and effectively localizes tobacco mosaic virus to sites of inoculation in transgenic tomato, as it does in tobacco. The ability to reconstruct the N-mediated resistance response to tobacco mosaic virus in tomato demonstrates the utility of using isolated resistance genes to protect crop plants from diseases, and it demonstrates that all the components necessary for N-mediated resistance are conserved in tomato.

Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution.

The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins.

We have isolated an Arabidopsis thaliana gene that codes for a receptor related to antifungal pathogenesis-related (PR) proteins. The PR5K gene codes for a predicted 665-amino acid polypeptide that comprises an extracellular domain related to the PR5 proteins, a central transmembrane-spanning domain, and an intracellular protein-serine/threonine kinase. The extracellular domain of PR5K (PR5-like receptor kinase) is most highly related to acidic PR5 proteins that accumulate in the extracellular spaces of plants challenged with pathogenic microorganisms. The kinase domain of PR5K is related to a family of protein-serine/threonine kinases that are involved in the expression of self-incompatibility and disease resistance. PR5K transcripts accumulate at low levels in all tissues examined, although particularly high levels are present in roots and inflorescence stems. Treatments that induce authentic PR5 proteins had no effect on the level of PR5K transcripts, suggesting that the receptor forms part of a preexisting surveillance system. When the kinase domain of PR5K was expressed in Escherichia coli, the resulting polypeptide underwent autophosphorylation, consistent with its predicted enzyme activity. These results are consistent with PR5K encoding a functional receptor kinase. Moreover, the structural similarity between the extracellular domain of PR5K and the antimicrobial PR5- proteins suggests a possible interaction with common or related microbial targets.

Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.

Two inducers of plant defense responses, 2,6-dichloroisonicotinec acid and salicylic acid, inhibit catalase activity in tobacco.

2,6-Dichloroisonicotinic acid (INA) and salicylic acid (SA) are potent inducers of plant defense responses including the synthesis of pathogenesis-related (PR) proteins and the development of enhanced disease resistance. A soluble SA-binding protein has been purified from tobacco with an affinity and specificity of binding that suggest it is a SA receptor. Recently, this protein has been shown to be a catalase whose enzymatic activity is inhibited by SA binding. We have proposed that the resulting increase in intracellular levels of reactive oxygen species plays a role in the induction of defense responses such as PR protein gene expression. Here we report that INA, like SA, binds the SA-binding protein/catalase and inhibits its enzymatic activity. In fact, the dose-response curves for inhibition of catalase by these two compounds are similar. Furthermore, the ability of both INA analogues and SA derivatives to bind and inhibit tobacco catalase correlates with their biological activity to induce PR-1 gene expression and enhance resistance to tobacco mosaic virus. Comparison of the structures of INA, SA, and their analogues reveals several common features that appear to be important for biological activity. Thus, these results not only suggest that INA and SA share the same mechanism of action that involves binding and inhibition of catalase but also further indicate an important role for reactive oxygen species in the induction of certain plant defense responses. This is supported by the demonstration that INA-mediated PR-1 gene activation is suppressed by antioxidants.

NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.

We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance. A. thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3. Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB). Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested. Interestingly, a hypersensitive-like response was still induced by several of the strains. The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica. Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III. Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

Induction, modification, and transduction of the salicylic acid signal in plant defense responses.

Studies in our laboratory as well as others strongly suggest that salicylic acid (SA) plays an important signaling role in plant defense against pathogens. We have found that increases in endogenous SA levels correlates with both resistance of tobacco to infection with tobacco mosaic virus and induction of defense-related genes such as that encoding pathogenesis-related protein 1 (PR-1). Some of this newly synthesized SA was conjugated to glucose to form SA beta-glucoside. A cell wall-associated beta-glucosidase activity that releases SA from this glucoside has been identified, suggesting that SA beta-glucoside serves as an inactive storage form of SA. By purifying a soluble SA-binding protein and isolating its encoding cDNA from tobacco, we have been able to further characterize the mechanism of SA signaling. This protein is a catalase, and binding of SA and its biologically active analogues inhibited catalase's ability to convert H2O2 to O2 and H2O. The resulting elevated levels of cellular H2O2 appeared to induce PR-1 gene expression, perhaps by acting as a second messenger. Additionally, transgenic tobacco expressing an antisense copy of the catalase gene and exhibiting depressed levels of catalase also showed constitutive expression of PR-1 genes. To further dissect the SA signaling pathway, we have tested several abiotic inducers of PR gene expression and disease resistance for their ability to stimulate SA production. Levels of SA and its glucoside rose following application of all of the inducers except 2,6-dichloroisonicotinic acid. 2,6-Dichloroisonicotinic acid was found to bind catalase directly and inhibit its enzymatic activity. Thus, it appears that many compounds that induce PR gene expression and disease resistance in plants inactivate catalases directly or indirectly.

The disease-resistance gene Pto and the fenthion-sensitivity gene fen encode closely related functional protein kinases.

Resistance to bacterial speck in tomato is governed by a gene-for-gene interaction in which a single resistance locus (Pto) in the plant responds to the expression of a specific avirulence gene (avrPto) in the pathogen. Disease susceptibility results if either Pto or avrPto are lacking from the corresponding organisms. Leaves of tomato cultivars that contain the Pto locus also exhibit a hypersensitive-like response upon exposure to an organophosphorous insecticide, fenthion. Recently, the Pto gene was isolated by a map-based cloning approach and was shown to be a member of a clustered multigene family with similarity to various protein-serine/threonine kinases. Another member of this family, termed Fen, was found to confer sensitivity to fenthion. The Pto protein shares 80% identity (87% similarity) with Fen. Here, Pto and Fen are shown to be functional protein kinases that probably participate in the same signal transduction pathway.

Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants.

Signal transduction in systemic acquired resistance.

Systemic acquired resistance (SAR) is an important component of plant defense against pathogen infection. Accumulation of salicylic acid (SA) is required for the induction of SAR. However, SA is apparently not the translocated signal but is involved in transducing the signal in target tissues. Interestingly, SA accumulation is not required for production and release of the systemic signal. In addition to playing a pivotal role in SAR signal transduction, SA is important in modulating plant susceptibility to pathogen infection and genetic resistance to disease. It has been proposed that SA inhibition of catalase results in H2O2 accumulation and that therefore H2O2 serves as a second messenger in SAR signaling. We find no accumulation of H2O2 in tissues expressing SAR; thus the role of H2O2 in SAR signaling is questionable.

Plant regeneration functional groups modulate the response to fire of soil enzyme activities in a Mediterranean shrubland

Soil enzymes are critical to soil nutrient cycling function but knowledge on the factors that control their response to major disturbances such as wildfires remains very limited. We evaluated the effect of fire-related plant functional traits (resprouting and seeding) on the resistance and resilience to fire of two soil enzyme activities involved in phosphorus and carbon cycling (acid phosphatase and β-glucosidase) in a Mediterranean shrublands in SE Spain. Using experimental fires, we compared four types of shrubland microsites: SS (vegetation patches dominated by seeder species), RR (patches dominated by resprouter species), SR (patches co-dominated by seeder and resprouter species), and IP (shrub interpatches). We assessed pre- and post-fire activities of the target soil enzymes, available P, soil organic C, and plant cover dynamics over three years after the fire. Post-fire regeneration functional groups (resprouter, seeder) modulated both pre- and post-fire activity of acid phosphatase and β-glucosidase, with higher activity in RR and SR patches than in SS patches and IP. However, we found no major differences in enzyme resistance and resilience between microsite types, except for a trend towards less resilience in SS patches. Fire similarly reduced the activity of both enzymes. However, acid phosphatase and β-glucosidase showed contrasting post-fire dynamics. While β-glucosidase proved to be rather resilient to fire, fully recovering three years after fire, acid phosphatase showed no signs of recovery in that period. Overall, the results indicate a positive influence of resprouter species on soil enzyme activity that is very resistant to fire. Long-lasting decrease in acid phosphatase activity probably resulted from the combined effect of P availability and post-fire drought. Our results provide insights on how plant functional traits modulate soil biochemical and microbiological response to fire in Mediterranean fire-prone shrublands.

Effects of plant functional traits on soil stability: intraspecific variability matters

This data set describes different vegetation, soil and plant functional traits (PFTs) of 15 plant species in 30 sampling plots of an agricultural landscape in the Haean-myun catchment in South Korea. We divided the data set into two main tables, the first one includes the PFTs data of the 15 studied plant species, and the second one includes the soil and vegetation characteristics of the 30 sampling plots. For a total of 150 individuals, we measures the maximum plant height (cm) and leaf size (cm**2), which means the leaf surface area for the aboveground compartment of each individual. For the belowground compartment, we measured root horizontal width, which is the maximum horizontal spread of the root, rooting length, which is the maximum rooting depth, root diameter, which is the average root diameter of a the whole root, specific root length (SRL), which is the root length divided by the root dry mass, and root/shoot ratio, which is the root dry mass divided by the shoot dry mass. At each of the 30 studied plots, we estimated three different variables describing the vegetation characteristics: vegetation cover (i.e. the percentage of ground covered by vegetation), species richness (i.e. the number of observed species) and root density (estimated using a 30 cm x 30 cm metallic frame divided into nine 10 cm x 10 cm grids placed on the soil profile), as we calculated the total number of roots that appear in each of the nine grids and then we converted it into percentage based on the root count, following. Moreover, in each plot we estimated six different soil variables: Bulk density (g/cm**3), clay % (i.e. percentage of clay), silt % (i.e. percentage of silt), soil aggregate stability, using mean weight diameter (MWD), penetration resistance (kg/cm**2), using pocket penetrometer and soil shear vane strength (kPa).

Fiji disease resistance in sugarcane: Relationship to cultivar preference in field populations of the planthopper vector Perkinsiella saccharicida

Populations of the planthopper vector Perkinsiella saccharicida on sugarcane cultivars resistant (cvs Q110 and Q87), moderately resistant (cvs Q90 and Q124) and susceptible (evs NCo310 and Q 102) to Fiji disease with known field resistance scores were monitored on the plant (2000-2001) and ratoon (2001-2002) crops. In both crops, the vector population remained very low, reaching its peak in the autumn. The vector population was significantly higher on cultivars susceptible to Fiji disease than on cultivars moderately resistant and resistant to Fiji disease. The number of R saccharicida adults, nymphs and oviposition sites per plant increased with the increase in the Fiji disease susceptibility. The results suggest that under low vector density, cultivar preference by the planthopper vector mediates Fiji disease resistance in sugarcane. To obtain resistance ratings in the glasshouse that reflect field resistance, glasshouse-screening trials should be conducted under both low and high vector densities, and the cultivar preference of the planthopper vector recorded along with Fiji disease incidence.

Identification of a major locus conferring resistance to powdery mildew (Erysiphe polygoni DC) in mungbean (Vigna radiata L. Wilczek) by QTL analysis

A major locus conferring resistance to the causal organism of powdery mildew, Erysiphe polygoni DC,, in mungbean (Vigna radiata L. Wilczek) was identified using QTL analysis with a population of 147 recombinant inbred individuals. The population was derived from a cross between 'Berken', a highly susceptible variety, and ATF 3640, a highly resistant line. To test for response to powdery mildew, F-7 and F-8 lines were inoculated by dispersing decaying mungbean leaves with residual conidia of E. polygoni amongst the young plants to create an artificial epidemic and assayed in a glasshouse facility. To generate a linkage map, 322 RFLP clones were tested against the two parents and 51 of these were selected to screen the mapping population. The 51 probes generated 52 mapped loci, which were used to construct a linkage map spanning 350 cM of the mungbean genome over 10 linkage groups. Using these markers, a single locus was identified that explained up to a maximum of 86% of the total variation in the resistance response to the pathogen.

Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1. function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.