Hari Krishnan | Cochin University of Science and Technology (original) (raw)
Papers by Hari Krishnan
Plant Science, 2000
A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified ... more A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised against the gel-purified protein. N-terminal sequence analysis identified the 53 kDa protein as the b-subunit of b-conglycinin. Results from Western blot analysis, using antibodies raised against the purified b-subunit of b-conglycinin, revealed that accumulation of this protein was enhanced in non-nodulating soybeans when the plants were supplemented with nitrogen. Results from Northern blot analysis indicate that non-nodulating soybeans in general had lower levels of mRNA for the major soybean seed proteins. A one-time application of nitrogen to non-nodulating soybeans enhanced the accumulation of the 1.6 kb b-conglycinin b-subunit mRNA. The mRNA levels of the 2.1 kb b-conglycinin a%-subunit and the 2.2 kb G4 glycinin in the non-nodulating soybeans were several-fold lower than in nodulating soybeans. Nitrogen application had no effect on the abundance of these RNA transcripts. The amount of RNA encoding an 8.5 kDa sulfur-rich protein was two-fold higher in non-nodulating soybeans when compared with nodulated soybeans. Nitrogen application reduced the abundance of this transcript to levels comparable with those of nodulated soybeans. Despite lower levels of the b-subunit of b-conglycinin, the methionine content of the total seed protein fraction was lower than that of nodulated soybeans. In contrast, non-nodulating soybeans contained more cysteine than nodulating soybeans.
![Research paper thumbnail of A Functional myoInositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean (Glycine max [L.] Merr](https://attachments.academia-assets.com/32116810/thumbnails/1.jpg)
](Journal of Bacteriology, 2001
Inositol derivative compounds provide a nutrient source for soil bacteria that possess the abilit... more Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.
Journal of Bacteriology, 2004
The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp... more The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid-and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.
Molecular Plant-microbe Interactions, 2005
Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, util... more Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretionminus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.
Molecular Plant-microbe Interactions, 2003
The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secreti... more The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.
Plant Science, 2000
A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified ... more A 53 kDa protein, which accumulates at low levels in non-nodulating Clark soybeans, was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised against the gel-purified protein. N-terminal sequence analysis identified the 53 kDa protein as the b-subunit of b-conglycinin. Results from Western blot analysis, using antibodies raised against the purified b-subunit of b-conglycinin, revealed that accumulation of this protein was enhanced in non-nodulating soybeans when the plants were supplemented with nitrogen. Results from Northern blot analysis indicate that non-nodulating soybeans in general had lower levels of mRNA for the major soybean seed proteins. A one-time application of nitrogen to non-nodulating soybeans enhanced the accumulation of the 1.6 kb b-conglycinin b-subunit mRNA. The mRNA levels of the 2.1 kb b-conglycinin a%-subunit and the 2.2 kb G4 glycinin in the non-nodulating soybeans were several-fold lower than in nodulating soybeans. Nitrogen application had no effect on the abundance of these RNA transcripts. The amount of RNA encoding an 8.5 kDa sulfur-rich protein was two-fold higher in non-nodulating soybeans when compared with nodulated soybeans. Nitrogen application reduced the abundance of this transcript to levels comparable with those of nodulated soybeans. Despite lower levels of the b-subunit of b-conglycinin, the methionine content of the total seed protein fraction was lower than that of nodulated soybeans. In contrast, non-nodulating soybeans contained more cysteine than nodulating soybeans.
Journal of Bacteriology, 2001
Inositol derivative compounds provide a nutrient source for soil bacteria that possess the abilit... more Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.
Journal of Bacteriology, 2004
The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp... more The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid-and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.
Molecular Plant-microbe Interactions, 2005
Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, util... more Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretionminus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.
Molecular Plant-microbe Interactions, 2003
The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secreti... more The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.