Modulation of defence and iron homeostasis genes in rice roots by the diazotrophic endophyte Herbaspirillum seropedicae.

Rice is staple food of nearly half the world's population. Rice yields must therefore increase to feed ever larger populations. By colonising rice and other plants, Herbaspirillum spp. stimulate plant growth and productivity. However the molecular factors involved are largely unknown. To further explore this interaction, the transcription profiles of Nipponbare rice roots inoculated with Herbaspirillum seropedicae were determined by RNA-seq. Mapping the 104 million reads against the Oryza sativa cv. Nipponbare genome produced 65 million unique mapped reads that represented 13,840 transcripts each with at least two-times coverage. About 7.4% (1,014) genes were differentially regulated and of these 255 changed expression levels more than two times. Several of the repressed genes encoded proteins related to plant defence (e.g. a putative probenazole inducible protein), plant disease resistance as well as enzymes involved in flavonoid and isoprenoid synthesis. Genes related to the synthesis and efflux of phytosiderophores (PS) and transport of PS-iron complexes were induced by the bacteria. These data suggest that the bacterium represses the rice defence system while concomitantly activating iron uptake. Transcripts of H. seropedicae were also detected amongst which transcripts of genes involved in nitrogen fixation, cell motility and cell wall synthesis were the most expressed.

Herbaspirillum rubrisubalbicans, a mild pathogen impairs growth of rice by augmenting ethylene levels.

KEY MESSAGE: Herbaspirillum rubrisubalbicans decreases growth of rice. Inoculation of rice with H. rubrisubalbicans increased the ACCO mRNA levels and ethylene production. The H. rubrisubalbicans rice interactions were further characterized by proteomic approach. Herbaspirillum rubrisubalbicans is a well-known growth-promoting rhizobacteria that can also act as a mild phyto-pathogen. During colonisation of rice, RT-qPCR analyses showed that H. rubrisubalbicans up-regulates the methionine recycling pathway as well as phyto-siderophore synthesis genes. mRNA levels of ACC oxidase and ethylene levels also increased in rice roots but inoculation with H. rubrisubalbicans impaired growth of the rice plant. A proteomic approach was used to identify proteins specifically modulated by H. rubrisubalbicans in rice and amongst the differentially expressed proteins a V-ATPase and a 14-3-3 protein were down-regulated. Several proteins of H. rubrisubalbicans were identified, including the type VI secretion system effector Hcp1, suggesting that protein secretion play a role colonisation in rice. Finally, the alkyl hydroperoxide reductase, a primary scavenger of endogenous hydrogen peroxide was also identified. Monitoring the levels of reactive oxygen species in the epiphytic bacteria by flow cytometry revealed that H. rubrisubalbicans is subjected to oxidative stress, suggesting that the alkyl hydroperoxide reductase is an important regulator of redox homeostasis in plant-bacteria interactions.

Metabolic profiling of two maize (Zea mays L.) inbred lines inoculated with the nitrogen fixing plant-interacting bacteria Herbaspirillum seropedicae and Azospirillum brasilense.

Maize roots can be colonized by free-living atmospheric nitrogen (N2)-fixing bacteria (diazotrophs). However, the agronomic potential of non-symbiotic N2-fixation in such an economically important species as maize, has still not been fully exploited. A preliminary approach to improve our understanding of the mechanisms controlling the establishment of such N2-fixing associations has been developed, using two maize inbred lines exhibiting different physiological characteristics. The bacterial-plant interaction has been characterized by means of a metabolomic approach. Two established model strains of Nif+ diazotrophic bacteria, Herbaspirillum seropedicae and Azospirillum brasilense and their Nif- couterparts defficient in nitrogenase activity, were used to evaluate the impact of the bacterial inoculation and of N2 fixation on the root and leaf metabolic profiles. The two N2-fixing bacteria have been used to inoculate two genetically distant maize lines (FV252 and FV2), already characterized for their contrasting physiological properties. Using a well-controlled gnotobiotic experimental system that allows inoculation of maize plants with the two diazotrophs in a N-free medium, we demonstrated that both maize lines were efficiently colonized by the two bacterial species. We also showed that in the early stages of plant development, both bacterial strains were able to reduce acetylene, suggesting that they contain functional nitrogenase activity and are able to efficiently fix atmospheric N2 (Fix+). The metabolomic approach allowed the identification of metabolites in the two maize lines that were representative of the N2 fixing plant-bacterial interaction, these included mannitol and to a lesser extend trehalose and isocitrate. Whilst other metabolites such as asparagine, although only exhibiting a small increase in maize roots following bacterial infection, were specific for the two Fix+ bacterial strains, in comparison to their Fix- counterparts. Moreover, a number of metabolites exhibited a maize-genotype specific pattern of accumulation, suggesting that the highly diverse maize genetic resources could be further exploited in terms of beneficial plant-bacterial interactions for optimizing maize growth, with reduced N fertilization inputs.

Comparative proteomics analysis of the rice roots colonized by Herbaspirillum seropedicae strain SmR1 reveals induction of the methionine recycling in the plant host.

Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant-bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDI-TOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots.