Multi locus sequence analysis and symbiotic characterization of novel Ensifer strains nodulating Tephrosia spp. in the Indian Thar Desert.

Phylogenetically diverse Ensifer strains associated with five species of Tephrosia growing in alkaline soils of semi-arid regions of the Thar Desert were characterized using multi locus sequence analysis. Based on 16S rRNA and four protein-coding housekeeping gene (recA, atpD, glnII and dnaK) sequences, the Tephrosia-Ensifer strains were genetically different from the type strains of Ensifer saheli, Ensifer kostiensis, Ensifer terangae (African origin) and Ensifer psoraleae (Asiatic origin). One strain, Ensifer sp. TL4, showed maximum similarity (99%) to Ensifer adhaerens LMG 20216T and formed a separate lineage close to it. Phylogenetic incongruence between sym and housekeeping genes was observed. The monophyletic origin of symbiotic genes from Asia in the Tephrosia-Ensifer strains from the Thar Desert suggests that they might have been acquired from a common ancestor and horizontally transferred. These novel strains are promiscuous, cross-nodulating some papilionoid crop species, mimosoid trees and the caesalpinioid Chamaecrista pumila. This study improves understanding of the distribution of Ensifer in unexplored and threatened alkaline arid regions of the Thar Desert and how this relates to other similar regions in the world.

Bacterial endophyte Sphingomonas sp. LK11 produces gibberellins and IAA and promotes tomato plant growth.

Plant growth promoting endophytic bacteria have been identified as potential growth regulators of crops. Endophytic bacterium, Sphingomonas sp. LK11, was isolated from the leaves of Tephrosia apollinea. The pure culture of Sphingomonas sp. LK11 was subjected to advance chromatographic and spectroscopic techniques to extract and isolate gibberellins (GAs). Deuterated standards of [17, 17-(2)H2]-GA4, [17, 17-(2)H2]-GA9 and [17, 17-(2)H2]-GA20 were used to quantify the bacterial GAs. The analysis of the culture broth of Sphingomonas sp. LK11 revealed the existence of physiologically active gibberellins (GA4: 2.97 ± 0.11 ng/ml) and inactive GA9 (0.98 ± 0.15 ng/ml) and GA20 (2.41 ± 0.23). The endophyte also produced indole acetic acid (11.23 ± 0.93 µM/ml). Tomato plants inoculated with endophytic Sphingomonas sp. LK11 showed significantly increased growth attributes (shoot length, chlorophyll contents, shoot, and root dry weights) compared to the control. This indicated that such phyto-hormones-producing strains could help in increasing crop growth.

Multilocus sequences confirm the close genetic relationship of four phytoplasmas of peanut witches'-broom group 16SrII-A.

Four witches'-broom diseases associated with Arachis hypogaea (peanut), Crotalaria pallida, Tephrosia purpurea, and Cleome viscosa were observed in Hainan Province, China during field surveys in 2004, 2005, and 2007. In previously reported studies, we identified these four phytoplasmas as members of subgroup 16SrII-A, and discovered that their 16S rRNA gene sequences were 99.9-100% identical to one another. In this study, we performed extensive phylogenetic analyses to elucidate relationships among them. We analyzed sequences of the 16S rRNA gene and rplV-rpsC, rpoB, gyrB, dnaK, dnaJ, recA, and secY combined sequence data from two strains each of the four phytoplasmas from Hainan province, as well as strains of peanut witches'-broom from Taiwan (PnWB-TW), "Candidatus Phytoplasma australiense", "Ca. Phytoplasma mali AT", aster yellows witches'-broom phytoplasma AYWB, and onion yellows phytoplasma OY-M. In the 16S rRNA phylogenetic tree, the eight Hainan strains form a clade with PnWB-TW. Analysis of the seven concatenated gene regions indicated that the four phytoplasmas collected from Hainan province cluster most closely with one another, but are closely related to PnWB-TW. The results of field survey and phylogenetic analysis indicated that Cr. pallida, T. purpurea, and Cl. viscosa may be natural plant hosts of peanut witches'-broom phytoplasma.

Y4lO of Rhizobium sp. strain NGR234 is a symbiotic determinant required for symbiosome differentiation.

Type 3 (T3) effector proteins, secreted by nitrogen-fixing rhizobia with a bacterial T3 secretion system, affect the nodulation of certain host legumes. The open reading frame y4lO of Rhizobium sp. strain NGR234 encodes a protein with sequence similarities to T3 effectors from pathogenic bacteria (the YopJ effector family). Transcription studies showed that the promoter activity of y4lO depended on the transcriptional activator TtsI. Recombinant Y4lO protein expressed in Escherichia coli did not acetylate two representative mitogen-activated protein kinase kinases (human MKK6 and MKK1 from Medicago truncatula), indicating that YopJ-like proteins differ with respect to their substrate specificities. The y4lO gene was mutated in NGR234 (strain NGROmegay4lO) and in NGR Omega nopL, a mutant that does not produce the T3 effector NopL (strain NGR Omega nopLOmegay4lO). When used as inoculants, the symbiotic properties of the mutants differed. Tephrosia vogelii, Phaseolus vulgaris cv. Yudou No. 1, and Vigna unguiculata cv. Sui Qing Dou Jiao formed pink effective nodules with NGR234 and NGR Omega nopL Omega y4lO. Nodules induced by NGR Omega y4lO were first pink but rapidly turned greenish (ineffective nodules), indicating premature senescence. An ultrastructural analysis of the nodules induced by NGR Omega y4lO revealed abnormal formation of enlarged infection droplets in ineffective nodules, whereas symbiosomes harboring a single bacteroid were frequently observed in effective nodules induced by NGR234 or NGR Omega nopL Omega y4lO. It is concluded that Y4lO is a symbiotic determinant involved in the differentiation of symbiosomes. Y4lO mitigated senescence-inducing effects caused by the T3 effector NopL, suggesting synergistic effects for Y4lO and NopL in nitrogen-fixing nodules.

Symbiosis-promoting and deleterious effects of NopT, a novel type 3 effector of Rhizobium sp. strain NGR234.

Establishment of symbiosis between certain host plants and nitrogen-fixing bacteria ("rhizobia") depends on type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS). Here, we report that the open reading frame y4zC of strain NGR234 encodes a novel rhizobial type 3 effector, termed NopT (for nodulation outer protein T). Analysis of secreted proteins from NGR234 and T3SS mutants revealed that NopT is secreted via the T3SS. NopT possessed autoproteolytic activity when expressed in Escherichia coli or human HEK 293T cells. The processed NopT exposed a glycine (G50) to the N terminus, which is predicted to be myristoylated in eukaryotic cells. NopT with a point mutation at position C93, H205, or D220 (catalytic triad) showed strongly reduced autoproteolytic activity, indicating that NopT is a functional protease of the YopT-AvrPphB effector family. When transiently expressed in tobacco plants, proteolytically active NopT elicited a rapid hypersensitive reaction. Arabidopsis plants transformed with nopT showed chlorotic and necrotic symptoms, indicating a cytotoxic effect. Inoculation experiments with mutant derivatives of NGR234 indicated that NopT affected nodulation either positively (Phaseolus vulgaris cv. Yudou No. 1; Tephrosia vogelii) or negatively (Crotalaria juncea). We suggest that NopT-related polymorphism may be involved in evolutionary adaptation of NGR234 to particular host legumes.

NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii.

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive- (Flemingia congesta, Tephrosia vogelii) or negative- (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram-negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGR(Omega)rhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.

Flavonoids, NodD1, NodD2, and nod-box NB15 modulate expression of the y4wEFG locus that is required for indole-3-acetic acid synthesis in Rhizobium sp. strain NGR234.

Flavonoids secreted by host plants activate, in conjunction with the transcriptional activator NodD, nod gene expression of rhizobia resulting in the synthesis of Nod factors, which trigger nodule organogenesis. Interestingly, addition of inducing flavonoids also stimulates the production of the phytohormone indole-3-acetic acid (IAA) in several rhizobia. Here, the molecular basis of IAA synthesis in Rhizobium sp. NGR234 was investigated. Mass spectrometric analysis of culture supernatants indicated that NGR234 is capable of synthesizing IAA via three different pathways. The production of IAA is increased strongly by exposure of NGR234 to daidzein in a NodD1-, NodD2-, and SyrM2-dependent manner. This suggests that the y4wEFG locus that is downstream of nod-box NB15 encodes proteins involved in IAA synthesis. Knockout mutations in y4wE and y4wF abolished flavonoid-inducible IAA synthesis and a functional y4wF was required for constitutive IAA production. The promoter activity of NB15 and IAA production both were enhanced by introduction of a multicopy plasmid carrying nodD2 into NGR234. Surprisingly, the y4wE mutant still nodulated Vigna unguiculata and Tephrosia vogelii, although the nodules contained less IAA and IAA conjugates than those formed by the wild-type bacterium.