Saul Burdman

Responses of agronomically important crops to inoculation with Azospirillum

Azospirilla are free-living rhizobacteria that are able to promote plant growth and increase yields in many crops of agronomic importance. It is assumed that the bacteria affect plant growth mainly by the production of plant growth promoting substances, which leads to an improvement in root development and an increase in the rate of water and mineral uptake. In the present review, we discuss the physiological responses of the plant roots to inoculation with Azospirillum, and report on field and greenhouse experiments carried out with these bacteria during 1994-2001 in Belgium, Uruguay, Mexico and Israel.

Surface Characteristics of Azospirillum brasilense in Relation to Cell Aggregation and Attachment to Plant Roots

The free-living bacteria of the genus Azospirillum live in close association with plant roots and represent one of the best-characterized plant growth promoting rhizobacteria (PGPR). The attachment of Azospirillum to the roots is essential for the establishment of an efficient association with the host plant. Azospirillum cells are able to aggregate under certain environmental conditions, leading to the formation of bacterial flocs. The bacterial surface plays an important role in the establishment of the bacteria-plant association as well as in the bacterial aggregation and data suggesting the involvement of extracellular polysaccharides and proteins in these phenomena have been published. This review summarizes the current knowledge on the involvement of surface components in the adhesion processes of Azospirillum. Emphasis is placed on A. brasilense, the species that has been the subject of most studies in the Azospirillum genus.

Aggregation in Azospirillum brasilense: effects of chemical and physical factors and involvement of extracellular components

A medium for consistent induction of aggregation of Azospirillum brasilense cells was developed and used to study the effects of chemical and physical factors as well as extracellular components involved in this phenomenon. Growth of A. brasilense strain Cd in a high C:N medium using fructose and ammonium chloride as C and N sources, respectively, resulted in flocculation visible to the naked eye after 24 h. No cell aggregates were formed after 72 h growth in low C:N medium. Aggregating cells, but not cells grown under low C:N, accumulated high amounts of poly-beta-hydroxybutyrate and the cell envelope contained a well-defined electron-dense layer outside the outer membrane. Suspending the aggregates in 0.2 or 0.5 M urea was the only treatment effective for disrupting aggregates. The concentration of exopolysaccharide produced by four different strains of A. brasilense, differing in their capacity to aggregate, strongly correlated with the extent of aggregation. Electrophoretic protein profiles from different fractions of aggregating and non-aggregating cells were compared. Differences were observed in the pattern of low-molecular-mass proteins and in the polar flagellin that has previously been proposed to be involved in adhesion processes. However, a mutant lacking both lateral and polar flagella showed the strongest aggregation. The involvement of polysaccharides and/or proteins in aggregation of A. brasilense is discussed.

Bacterial Genes Involved in Type I Secretion and Sulfation Are Required to Elicit the Rice Xa21-Mediated Innate Immune Response

Innate immunity to microorganisms relies on the specific sensing of pathogen-associated molecules by host recognition receptors. Whereas studies in animals have largely focused on the recognition of extracellular pathogen-associated molecules by the TLR (toll-like receptor) superfamily, few studies have been carried out in plants, and it is not understood how these molecules are secreted or modified. The rice Xa21 gene encodes a receptor-like kinase that provides immunity against strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae carrying AvrXa21 activity. We identified four X. oryzae pv. oryzae genes that are required for AvrXa21 activity. raxA, raxB, and raxC encode proteins with similarity to a membrane fusion protein, an ATP-binding cassette transporter, and an outer membrane protein, respectively, of bacterial type I secretion systems. The fourth gene, raxST, encodes a sulfotransferase-like protein. Sequence analysis of three naturally occurring X. oryzae pv. oryzae strains no longer recognized by Xa21 revealed alterations in the raxST and raxA genes. The raxC gene complemented an Escherichia coli tolC mutant for secretion of a double glycine-leader peptide confirming the function of raxC in type I secretion. These results indicate that bacterial type I secretion is necessary for Xa21-mediated recognition and immunity and further suggest that type I secretion and modification of pathogen-associated molecules play an important role in triggering the innate immune response in rice.

GLYCOALKALOID METABOLISM1 Is Required for Steroidal Alkaloid Glycosylation and Prevention of Phytotoxicity in Tomato

Steroidal alkaloids (SAs) are specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. In this study, the role of GLYCOALKALOID METABOLISM1 (GAME1) in the biosynthesis of tomato SAs was revealed, highlighting the importance of GAME1 in SA glycosylation and in reducing the toxicity of SA metabolites to the plant cell. Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

Effects of Azospirillum brasilense on nodulation and growth of common bean (Phaseolus vulgaris L.)

Abstract Combined inoculation of potted common bean plants with Rhizobium and Azospirillum significantly increased both upper and total nodule number and N2-fixation as compared with inoculation with Rhizobium alone. At an Azospirillum concentration of 108 cfu ml−1, the combined inoculation reduced root and shoot dry weight accumulation in comparison with Rhizobium alone and with uninoculated controls. However, when the combined inoculation was performed using a lower Azospirillum concentration (5 × 106 cfu ml−1), positive effects on plant growth were observed, although the enhancement of nodulation and N2-fixation were not as great as observed at the higher Azospirillum concentration. Field application of these findings are discussed.

Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato

A group of plant proteins, expansins, have been identified as wall-loosening factors and as facilitators of cell expansion in vivo. The root knot nematode Meloidogyne javanica establishes a permanent feeding site composed of giant cells surrounded by gall tissue. We used quantitative PCR and in situ localization to demonstrate the induction of a tomato (Lycopersicon esculentum cv. VF36) expansin (LeEXPA5) expression in gall cells adjacent to the nematode feeding cells. To further characterize the biological role of LeEXPA5 we have generated LeEXPA5-antisense transgenic roots. The ability of the nematode to establish a feeding site and complete its life cycle, the average root cell size and the rate of root elongation were determined for the transgenic roots, as well as the level of LeEXPA5 expression in non-infected and nematode-infected roots. Our results demonstrated that a decrease of LeEXPA5 expression reduces the ability of the nematode to complete its life cycle in transgenic roots. We suggest that a plant-originated expansin is necessary for a successful parasitic nematode–plant interaction.

Molecular, physiological, and host-range characterization of Acidovorax avenae subsp. citrulli isolates from watermelon and melon in Israel.

Bacterial fruit blotch (BFB), caused by Acidovorax avenae subsp. citrulli, is a serious disease of cucurbit plants. The first important occurrence of BFB in Israel was during 2000 to 2003 on watermelon and melon. Twelve bacterial isolates associated with these outbreaks were confirmed as A. avenae subsp. citrulli by pathogenicity assays, gas chromatography of fatty-acid methyl esters, and substrate-utilization profiles. The isolates were characterized in terms of their aggressiveness in different hosts by seed, seedling, and fruit inoculations, and according to their DNA fingerprinting profiles using pulse-field gel electrophoresis (PFGE) and repetitive-PCR approaches. Results from the present work agree with previous studies supporting the existence of two differentiated groups within A. avenae subsp. citrulli, one including strains that are more associated with watermelon (group II), the other consisting of strains that are usually associated with nonwatermelon cucurbits (group I). This study indicates that isolates from both groups have been introduced to Israel. PFGE analysis revealed that the 12 analyzed isolates can be divided into five different haplotypes, of which four were previously unreported. Additional differentiating features between group I and II strains are presented.

RaxH/RaxR: A Two-Component Regulatory System in Xanthomonas oryzae pv. oryzae Required for AvrXa21 Activity

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, one of the most serious diseases in rice. X. oryzae pv. oryzae Philippine race 6 (PR6) strains are unable to establish infection in rice lines expressing the resistance gene Xa21. Although the pathogen-associated molecule that triggers the Xa21-mediated defense response (AvrXa21) is unknown, six rax (required for AvrXa21 activity) genes encoding proteins involved in sulfur metabolism and Type I secretion were recently identified. Here, we report on the identification of two additional rax genes, raxR and raxH, which encode a response regulator and a histidine protein kinase of two-component regulatory systems, respectively. Null mutants of PR6 strain PXO99 that are impaired in either raxR, raxH, or both cause lesions significantly longer and grow to significantly higher levels than does the wild-type strain in Xa21-rice leaves. Both raxR and raxH mutants are complemented to wild-type levels of AvrXa21 activity by introduction of expression vectors carrying raxR and raxH, respectively. These null mutants do not affect AvrXa7 and AvrXa10 activities, as observed in inoculation experiments with Xa7- and Xa10-rice lines. Western blot and raxR/gfp promoter-reporter analyses confirmed RaxR expression in X. oryzae pv. oryzae. The results of promoter-reporter studies also suggest that the previously identified raxSTAB operon is a target for RaxH/RaxR regulation. Characterization of the RaxH/RaxR system provides new opportunities for understanding the specificity of the X. oryzae pv. oryzae-Xa21 interaction and may contribute to the identification of AvrXa21.

GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.