A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes.

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.     

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.     

Sinorhizobium fredii HH103 rkp-3 genes are required for K-antigen polysaccharide biosynthesis, affect lipopolysaccharide structure and are essential for infection of legumes forming determinate nodules

The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule-forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule-forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS.     

Sinorhizobium fredii HH103 does not strictly require KPS and/or EPS to nodulate Glycyrrhiza uralensis, an indeterminate nodule-forming legume

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.     

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.     

Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean

Glycine max (soybean) plants can be nodulated by fast-growing rhizobial strains of the genus Sinorhizobium as well as by slow-growing strains clustered in the genus Bradyrhizobium. Fast-growing rhizobia strains with different soybean cultivar specificities have been isolated from Chinese soils and from other geographical regions. Most of these strains have been clustered into the species Sinorhizobium fredii. The S. fredii strain HH103 was isolated from soils of Hubei province, Central China and was first described in 1985. This strain is capable to nodulate American and Asiatic soybean cultivars and many other different legumes and is so far the best studied fast-growing soybean-nodulating strain. Additionally to the chromosome S. fredii HH103 carries five indigenous plasmids. The largest plasmid (pSfrHH103e) harbours genes for the production of diverse surface polysaccharides, such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS). The second largest plasmid (pSfrHH103d) is a typical symbiotic plasmid (pSym), carrying nodulation and nitrogen fixation genes. The present mini review focuses on symbiotic properties of S. fredii HH103, in particular on nodulation and surface polysaccharides aspects. The model strain S. fredii HH103 was chosen for genomic sequencing, which is currently in progress. First analyses of the draft genome sequence revealed an extensive synteny between the chromosomes of S. fredii HH103 and Rhizobium sp. NGR234.     

接种量对费氏中华根瘤菌HN01DL根圈定殖与结瘤的影响

在灭菌土和未灭菌土盆栽系统中 ,研究了大豆种子的表面初始接种量对费氏中华根瘤菌HN0 1DL在大豆根圈中的定殖动态与结瘤的影响。结果表明 ,与 3个接种量对应的早期根圈定殖动态和水平有明显差异 ,但随着宿主植物根系的生长其差异逐渐减小 ,整个定殖动态曲线的变化和定殖密度趋向一致 ,并且发现 3个不同的初始接种量对HN0 1DL在大豆黑农 33根系上的结瘤数量和占瘤率没有显著影响。     

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.     

Sex allocation and mating systems in pigeonpea,Cajanus cajan (Fabaceae)

Sex allocation theory predicts that: (1) resources allocated to androecium should decrease with an increase in selfing, (2) a decrease in androecium biomass should be accompanied by an increase in the biomass of pistils, and (3) a decrease in androecium biomass should be coupled with a decrease in flower size, specifically corolla biomass. Another predicted change in reproductive traits associated with variation in selfing concerns seed to ovule ratios, but does not directly stem from sex allocation theory. It has been postulated that seed to ovule ratios should be positively correlated with the amount of selfing. These predictions were tested for six accessions of pigeonpea,Cajanus cajan L., that differed in selfing rates. The results were remarkably in accordance with the predictions. We conclude that sex allocation theory provides a powerful tool to understand the evolution of many reproductive traits in plants.     

gurke and pasticcino3 mutants affected in embryo development are impaired in acetyl-CoA carboxylase

Normal embryo development is required for correct seedling formation. The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry. We have recently identified lethal acc1 mutants affected in acetyl-CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype. A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles. The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast. Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl-CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.     

Evaluation of Cajanus cajan (pigeon pea) for phytoremediation of landfill leachate containing chromium and lead

Landfill leachates containing heavy metals are important contaminants and a matter of great concern due to the effect that they might have on ecosystems. We evaluated the use of Cajanus cajan to remove chromium and lead from landfill leachates. Eight-week-old plants were submitted to varied tests to select the experimental conditions. Water assays with a solution (pH 6) containing leachate (25% v/v) were selected; the metals were added as potassium dichromate and lead (II) nitrate salts. Soil matrices that contained leachate (30% v/v) up to field capacity were used. For both water and soil assays, the metal concentrations were 10 mg kg^?1. C. cajan proved able to remove 49% of chromium and 36% of lead, both from dilute leachate. The plants also removed 34.7% of chromium from irrigated soil, but were unable to decrease the lead content. Removal of nitrogen from landfill leachate was also tested, resulting in elimination of 85% of ammonia and 70% of combined nitrite/nitrate species. The results indicate that C. cajan might be an effective candidate for the rhizofiltration of leachates containing chromium and lead, and nitrogen in large concentrations.     

Genetic diversity studies and identification of SSR markers associated with Fusarium wilt (Fusarium udum) resistance in cultivated pigeonpea (Cajanus cajan)

Genetic diversity and identification of simple sequence repeat markers correlated with Fusarium wilt resistance was performed in a set of 36 elite cultivated pigeonpea genotypes differing in levels of resistance to Fusarium wilt. Twenty-four polymorphic sequence repeat markers were screened across these genotypes, and amplified a total of 59 alleles with an average high polymorphic information content value of 0.52. Cluster analysis, done by UPGMA and PCA, grouped the 36 pigeonpea genotypes into two main clusters according to their Fusarium wilt reaction. Based on the Kruskal–Wallis ANOVA and simple regression analysis, six simple sequence repeat markers were found to be significantly associated with Fusarium wilt resistance. The phenotypic variation explained by these markers ranged from 23.7 to 56.4%. The present study helps in finding out feasibility of prescreened SSR markers to be used in genetic diversity analysis and their potential association with disease resistance.     

Evidence for Cytokinin Involvement in Rhizobium (IC3342)-Induced Leaf Curl Syndrome of Pigeonpea (Cajanus cajan Millsp.)

A uniquely abnormal shoot development (shoot tip-bending, leaf curling, release from apical dominance, and stunted growth) in pigeonpea (Cajanus cajan Millsp) induced by a nodulating Rhizobium strain, IC3342, is thought to be due to a hormonal imbalance. Amaranthus betacyanin bioassay indicated that xylem exudate and leaf extracts from pigeonpea plants with Rhizobium-induced leaf curl symptoms contained high concentrations of cytokinin relative to those in normal plants. Radioimmunoassay (RIA) of samples purified with high performance liquid chromatography revealed that zeatin riboside (ZR) and dihydrozeatin riboside (DZR) concentrations in xylem sap from plants with leaf curl symptoms were 7 to 9 times higher than those in the sap from symptomless, nodulated plants. The sap from symptomless plants nodulated by a Curl⁻ mutant had ZR and DZR concentrations comparable to those in the normal plant sap. RIA indicated that the respective concentrations of zeatin and N⁶-isopenteny-ladenine in culture filtrates of the curl-inducing strain IC3342 were 26 and 8 times higher than those in filtrates of a related normal nodulating strain (ANU240). Gas chromatographic-mass spectrometric analyses revealed similar differences. Gene-specific hybridization and sequence comparisons failed to detect any homology of IC3342 DNA to Agrobacterium tumefaciens or Pseudomonas savastanoi genetic loci encoding enzymes involved in cytokinin biosynthesis.     

Diurnal variations in leaf water potential and stomatal conductance of pigeon pea (Cajanus cajan (L.) Millsp.) cultivars as affected by irrigation levels

Two cultivars of pigeon pea (Cajanus cajan (L.)Millsp.) UPAS-120 and Parbhat were grown in the field under two irrigation treatments: no irrigation and irrigation when cumulative pan evaporation was equal to 50 per cent depletion of available water content in one metre root zone depth. Diurnal changes in leaf water potential and stomatal conductance were recorded on two daysi.e. October 1, 1979 and October 28, 1979 which corresponded to reproductive growth stage of the crop. Plant water potential decreased rapidly during the day up to about 15.00 and increased during evening hours. Higher leaf water potential was recorded in irrigated treatment on both dates. Adaxial and abaxial stomata differed in their response to water stress. Adaxial stomatal conductance started declining during early morning hours, however, abaxial conductance firstly increased up to 09.00 then decreased and increased again in the afternoon except in irrigated crop of cv. UPAS-120 on 28th October, where conductance never increased after 09.00. The reduced rate of stomatal conductance during day hours may be identified as one of the characteristics responsible for drought tolerance in pigeon pea.     

Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed     

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.     

Ontogenetic Changes in the Level of Ureides and Enzymes of their Metabolism in Various Plant Parts of Pigeonpea (Cajanus cajan)

Levels of allantoin and allantoic acid in shoots, roots, nodulesand leaves of pigeonpea plant, in general, followed the patternof acetylene reduction in nodules, increasing progressivelyfrom 15 days after sowing (DAS) and attaining peaks at 75 DASand 60 DAS, respectively, except in shoots where their contentsevinced maximum values at pod-setting (90 DAS). Activity ofGS in nodules and shoots reached a maximum at 60 DAS and 75DAS, respectively. However, in leaves and roots, the enzymeshowed a biphasic behaviour with peaks at days 60 and 105 inleaves and at days 75 and 105 in roots. GDH activity in nodulespeaked at 60 DAS, whereas, in leaves and roots, the maximumactivity was observed at flowering (75 DAS). Uricase was presentonly in nodules with peak activity at flowering. Allantoinaseactivity again peaked at flowering, where nodules had maximumactivity followed by leaves, roots and shoots. Urease couldbe detected in all the organs with maximum activity at 60 DASin leaves followed by roots and nodules. Except uricase, allthe enzymes reported above were also present in reproductivestructures. Compared to GS, GDH was more active both in flowerbuds and developing pods. Seeds, compared to podwalls, containedhigher activities of GDH, allantoinase and urease at day 105.Only allantoin could be detected in seeds and podwalls at day105. Key words: Cajanus cajan, Allantoin, Allantoic acid, Nitrogenase, Glutamine synthetase, Glutamate dehydrogenase, Uricase, Allantoinase, Urease, Development