Defense and counterdefense in the plant world

An important role of RNA interference (RNAi)-like pathways in plants is defense against viral infection. Viruses can overcome this defense by expressing proteins that suppress the pathway. A new study of Agrobacterium tumefaciens infection reveals that this plant pathogen, although a bacterium, also induces and then suppresses the host RNAi response. © 2006 Nature Publishing Group.

Exploring plant genomes by RNA-induced gene silencing

The nucleotide sequences of several animal, plant and bacterial genomes are now known, but the functions of many of the proteins that they are predicted to encode remain unclear. RNA interference is a gene-silencing technology that is being used successfully to investigate gene function in several organisms - for example, Caenorhabditis elegans. We discuss here that RNA-induced gene silencing approaches are also likely to be effective for investigating plant gene function in a high-throughput, genome-wide manner.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system.

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

Evidence for VirB4-mediated dislocation of membrane-integrated VirB2 pilin during biogenesis of the Agrobacterium VirB/VirD4 type IV secretion system.

Agrobacterium VirB2 pilin is required for assembly of the VirB/VirD4 type IV secretion system (T4SS). The propilin is processed by signal sequence cleavage and covalent linkage of the N and C termini, and the cyclized pilin integrates into the inner membrane (IM) as a pool for assembly of the secretion channel and T pilus. Here, by use of the substituted cysteine accessibility method (SCAM), we defined the VirB2 IM topology and then identified distinct contributions of the T4SS ATPase subunits to the pilin structural organization. Labeling patterns of Cys-substituted pilins exposed to the membrane-impermeative, thiol-reactive reagent 3-(N-maleimidopropionyl)biocytin (MPB) supported a topology model in which two hydrophobic stretches comprise transmembrane domains, an intervening hydrophilic loop (residues 90 to 94) is cytoplasmic, and the hydrophilic N and C termini joined at residues 48 and 121 form a periplasmic loop. Interestingly, the VirB4 ATPase, but not a Walker A nucleoside triphosphate (NTP) binding motif mutant, induced (i) MPB labeling of Cys94, a residue that in the absence of the ATPase is located in the cytoplasmic loop, and (ii) release of pilin from the IM upon osmotic shock. These findings, coupled with evidence for VirB2-VirB4 complex formation by coimmunoprecipitation, support a model in which VirB4 functions as a dislocation motor to extract pilins from the IM during T4SS biogenesis. The VirB11 ATPase functioned together with VirB4 to induce a structural change in the pilin that was detectable by MPB labeling, suggestive of a role for VirB11 as a modulator of VirB4 dislocase activity.

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions.

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.

The development of a sustainable agricultural industry through the application of biochar and pyroligneous acid

This paper marks the first in a series of studies into the potential use of pyrolysis products in the development of more sustainable practices within the agricultural industry. In this study, the immediate benefits of the application of biochar to crop yields of Raphanus sativus (radishes) are assessed. Furthermore, the study reports on the preliminary findings into the potential application of pyroligneous acid (wood vinegar) as a biocidal agent against crop disease. Although germination tests undertaken on biochar/compost blends of up to 1: 2, by weight, showed no significant adverse effect from the addition of the nutrient rich carbonaceous solid, evidence of substantial increases in crop yield through the addition of biochar were not observed. In sharp contrast, zones of inhibition were observed at 3-10 vol. % upon application of pyroligneous acid to two causal agents responsible for certain diseases in vegetable and fruit crops, i.e. Rhizobium radiobacter (agrobacterium tumefaciens) and Xanthomonas campestris, highlighting the versatility in the application of pyrolysis products and avenues for exploration in the development of this biomass conversion technology.

National Rhizobium Program - Managing rhizobia to maximise Nitrogen fixation

Inoculation of legumes with rhizobia is fundamental to sustainable productivity of Australian agriculture. The National Rhizobium Program has specific aims of sustaining and increasing Nitrogen fixation by legumes in Australian agriculture.

Purification and properties of an NADP+-specific isocitrate dehydrogenase from Rhizobium meliloti

An NADP+-specific isocitrate dehydrogenase has been purified and characterized from Rhizobium meliloti. The enzyme showed Mn++ or Mg++ requirement. The apparent Km values were 2.00×10-5 m and 1.51×10-5 m for dl-isocitrate and NADP+, respectively. The enzyme was inhibited by ATP, to a lesser extent by ADP and AMP. agr-Ketoglutarate also inhibited the enzyme activity. Oxalacetate and glyoxylate together inhibited the enzyme activity. The inhibition was competitive. Studies with thiol inhibitors suggested that the enzyme contained a sulfhydryl group at or near the active site. The enzyme has an approximate molecular weight of 60 000. Fluorescence studies suggested that the enzyme contained tryptophan.

Differential binding of peanut agglutinin with lipopolysaccharide of homologous and heterologous Rhizobium

To establish the crucial role of lipopolysaccharide in the initial recognition event of symbiotic peanut-Rhizobium system the ability of various surface polysaccharides isolated from Bradyrhizobium arachis to inhibit the precipitin reaction between peanut agglutinin and asialoganglioside: deoxycholate (1:1) micelles was estimated. It was compared with that of nonsymbiotic systems e.g. Bradyrhizobium japonicum, Bradyrhizobium ciceris and Escherichia coli. Peanut agglutinin was found to interact more strongly with the lipopolysaccharide of Bradyrhizobium arachis than the exopolysaccharide or capsular polysaccharide. The inhibitory capacity of lipopolysaccharides from homologous and heterologous Bradyrhizobium as measured in terms of the concentration necessary for 50 percent inhibition of precipitin reaction were 1428, 500, 410, and 277 times less than that of lactose for Bradyrhizobium arachis, B. japonicum, B. ciceris and Escherichia coli, respectively. These results support that host lectin peanut agglutinin can recognize homologous Bradyrhizobium lipopolysaccharide by virtue of its binding specificity of higher magnitude.

Evaluación de cinco cepas de Rhizobium leguminosarum bv. phaseoli en el cultivo del frijol (Phaseolus vulgaris L.) var. Rev. 84

Esta investigación se realizó en el Centro Experimental "La Compañía" ubicada en San Marcos, Carazo, con el objetivo de evaluar la adaptabilidad de las cepas en las condiciones estudiadas, determinar cuál de las cepas es la más infectiva y efectiva en cuanto a la nodulación y determinar su efecto en el crecimiento y rendimiento del cultivo del frijol (Phaseolus vulgaris L.) var. Rev. 84. Los factores evaluados fueren 5 cepas de Rhisoblum leguminosearum by. phaseoli y 3 testigos: 1 Testigo Absoluto (no inoculado. no fertilizado) Y 2 Testigos Relativos (no inoculados y fertilizados). El diseño experimental utilizado fue el de bloques completos al azar (SCA), con 4 repeticiones. La parcela experimental del ensayo fue de 32 (8 m x 4 m), la distancia de siembra fue de 0.1 m entre plantas y 0.4 m entre surcos. Las variables analizadas fueron: número; eficiencia y peso seco de nódulos por planta, peso seco de área foliar y rendimiento. Los datos se procesaron usando análisis de varianza (AHUEVA) y se utilizó la prueba de rangos múltiples de DUNCAN (P ≤ 0. 05). No se observó diferencias significativas para las variables de nodulación y rendimiento, determinándose efecto significativo sólo para el peso seco de área foliar en la I y II etapa de evaluación Sin embargo, se determinó quo la cepa CR-436 fue la que presentó mayor infectividad en el sistema radicular y la cepa CIAT-899, la que presentó el mejor porcentaje de efectividad nodular y con la que se obtuvo el mayor promedio de peso seco de área foliar. El mejor rendimiento fue obtenido con la cepa CR-436.

Incremento de la productividad de frijol (Phaseolus vulgaris L.) a nivel de finca mediante la inoculación con Rhizobium leguminosarum bv. phaseoli

El efecto de una mezcla de tres cepas de Rhizobium leguminosarum biovar phaseoll (CIAT-613, CR-477 y KIM-5) relacionado con los factores limitantes del suelo (P, Ca, Cu, y Zn) sobre la simbiosis en tres variedades de Phaseolus vulgaris L. (DOR-364, ESTELI-90B y FIEVOLUCION-84) fue estudiado en un suelo Molisol y un Aluvial en las localidades de San Diego (Nandaime) y San Lorenzo (La Trinidad, Estelí), respectivamente, bajo condiciones de labranza convencional para ambas localidades. En la localidad de San Lorenzo se trabajó con las variedades DOR-364 y EST-908 con la corrección del cobre (factor A) y el zinc (factor 8), mientras que en la localidad de San Diego, el trabajo se realizó con las variedades DOR-364 y REVOLUCION-84 y la corrección del calcio (factor A) y el fósforo (factor 8), Ambos estudios se llevaron a cabo en época de postrera de 1994. En los dos ensayos la inoculación se hizo directamente a la semilla. Los tratamientos a evaluar fueron los siguientes: Alto nitrógeno, bajo nitrógeno como testigos (sin inocular), mezcla de inoculantes con (-A,-B), (+A,-B), (-A,+B), y (+A,+B) para un total de seis tratamientos por cada variedad. El diseño usado fue de bloques completos al azar (B.C.A). Las variables evaluadas fueron: Número y peso seco de nódulos, peso de materia seca de la planta en R6, peso de mil granos y rendimiento de grano en R9. Los datos se procesaron usando análisis de varianza (ANDEVA) y se utilizó la prueba de rangos múltiples de DUNCAN (P ≤ 0,05). Se observó a nivel general en ambos experimentos que la variedad introducida mostró un mayor rendimiento y que el mejor rendimiento de grano se obtuvo con el tratamiento en el que se usó alta dosis de nitrógeno y sin inocular. Por otro lado el rendimiento de frijol fue afectado negativamente por las aplicaciones de zinc en el caso del experimento en San Lorenzo. Para la localidad de San Diego se obtuvo respuesta positiva a las aplicaciones de fósforo junto con la mezcla de inoculantes usados. En los dos experimentos no se encontró respuesta significativa con el uso de la mezcla de inoculantes relacionados con los elementos limitantes del suelo comparado con los testigos.