Diversity of plant growth-promoting bacteria associated with sugarcane

The sugarcane (Saccharum spp) presents economic importance, mainly for tropical regions, being an important Brazilian commodity. However, this crop is strongly dependent on fertilizers, mainly nitrogen (N). This study assessed the plant growth-promoting bacteria (PGPB) associated with sugarcane that could be used as a potential inoculant to the crop. We evaluated the genetic diversity of PGPB in the plant tissue of sugarcane varieties (RB 867515, RB 1011, and RB 92579). The primer BOX-A1R was used to differentiate the similar isolated and further sequencing 16S rRNA ribosomal gene. The 16S rRNA gene showed the presence of seven different genera distributed into four groups, the genus Bacillus, followed by Paenibacillus (20%), Burkholderia (14%), Herbaspirillum (6%), Pseudomonas (6%), Methylobacterium (6%), and Brevibacillus (3%). The molecular characterization of endophytic isolates from sugarcane revealed a diversity of bacteria colonizing this plant, with a possible biotechnological potential to be used as inoculant and biofertilizers

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. [Int Microbiol 2006; 9(2):125-133

NrcR, a new transcriptional regulator of Rhizobium tropici CIAT 899 involved in the Legume root-nodule symbiosis.

The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wildtype strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species

Draft genome sequence of Pseudomonas fluorescens strain ET76, isolated from rice Rhizosphere in Northwestern Morocco.

Pseudomonas fluorescens ET76 was isolated from rice rhizosphere in northwestern Morocco. Its draft genome was estimated to be 6,681,652 bp with 5,789 coding sequences (CDSs). Genes encoding for type I to VI secretion systems, PvdQ, proteases, siderophores, hydrogen cyanide synthase, ACC-deaminase, among others, highlight its potential use in biological control of plant pathogens.201

Unraveling plant cellular targets for the Rhizobium-specific effectors NopL and NopP

Resumen del póster presentado al 12th European Nitrogen Fixation Conference, celebrado en Budapest (Hungria) del 25 al 28 de agosto de 2016.Pathogenic Gram-negative use a specialized apparatus called Type 3 secretion system (T3SS) to deliver effectors directly into the eukaryotic host cells. These effectors suppress plant defenses to promote disease but they can also be recognized by specific plant receptors that trigger a strong defense reaction to eliminate the pathogen. The T3SS has also been found in some symbiotic rhizobial strains and the effectors secreted are involved in host-range determination and symbiotic efficiency. The broad host-range bacterium S. fredii HH103 secretes two proteins through the T3SS, NopL and NopP, which are specific for rhizobia. In this work we studied the function of both effectors in the symbiosis with soybean, which is considered its natural host plant. NopL and NopP were phosphorylated by soybean root kinases and the phosphorylation cascade was Ca2+ - and calmodulin-dependent. While the signaling pathway that culminates in the phosphorylation of NopL included ser/thr and MAPKK kinases, in the case of NopP this pathway involved ser/thr and tyr kinases but not MAPKK kinases. Transient expression of both nopL and nopP fused to YFP in Nicotiana benthamiana leaves and further confocal imaging indicated that they localized to the nucleus of the host cell. The use of a yeast-based array to determine possible effectors functions indicated that NopP could be involved in nuclear localization and migration. Finally, co-immunoprecipitation analyses of N. benthamiana NopL- and NopP-interacting proteins and further mass spectrometry analyses identified several potential plant targets for these effectors. The most interesting interactions are currently being validated by Bimolecfular Fluorescence Complementation (BiFC).This work was supported by project P11-CVI-7050 of the Junta de Andalucía.Peer Reviewe

Genome of Rhizobium leucaenae strains CFN 299T and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions.

Background: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici “group” are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian “Cerrados” soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299T and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions. Results: The genomes of R. leucaenae strains CFN 299T and CPAO 29.8 were estimated at 6.7–6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299T is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different. Conclusion: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899T and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.CNPq (National Council for Scientific and Technological Development)Science without Borders (400205/2012-5)Ministerio de Economía y Competitividad (FPU14_00160

Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899.

Transcription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes?five?and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants. The three nodD mutants were built by insertion of ? interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.). Phenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-geneinducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions.201