In order to visualize bacterial gene expression in the plant, we used transcriptional fusions of target genes such as nifH, one of the structural genes for the nitrogenase complex reducing N2 to ammonia, with the reporter gene gfp (encoding the jellyfish green fluorescent protein) or gus (encoding p-glucuronidase). Infection studies using Azoarcus sp. BH72 on rice seedlings in gnotobiotic culture revealed, that bacterial nifH::gfp is expressed at high levels inside the roots (Reinhold-Hurek, Hurek
1998). The sites of nitrogenase gene expression are the infection sites at emerging lateral roots and root tips as well as microcolonies inside the cortex in the aerenchymatic air spaces (Egener et al.
1999). It is remarkable that this endophytic gene expression occurs in the apoplast, apparently depending on apoplastic nutrient flow. Only minor amounts of carbon source (5 mg of malate per 1) were added as "starter" to the plant medium, thus the endophytic nifH gene expression appears to be driven by carbon- and energy sources supplied by the plant. In contrast, in other systems studied such as Gluconacetobacter diazotrophicus in sugar cane (James, Olivares 1998), significant nif gene expression was only detected in gnotobiotic systems when high amounts of carbon source were added to the plant medium.
Since nifH gene expression in Azoarcus sp. BH72 is similarly regulated in response to O2 and ammonium as the nitrogenase activity is (Hurek et al. 1994), the reporter gene studies demonstrate that the rice root apoplast may provide a suitable microhabitat for endophytic nitrogen fixation of Azoarcus sp. BH72. This is also the case for the host plant from which this strain was originally isolated, Kallar grass (Reinhold et al. 1986). It was shown by in situ hybridization studies that field-grown plants harbor Azoarcus sp. nifH mRNA in the aerenchyma (Hurek et al. 1997).
Recent findings suggest that the assumption that diazotrophic endophytes do not cause damage of plants cannot be generalized. Previously, a phytopathogenic species closely related to Herbaspirillum seropedicae, H. rubrisubalbicans, was detected to cause mild symptoms of disease in leafs of certain sugar cane cultivars, in others it grew as a non-pathogenic endophyte (Olivares et al. 1997). In our laboratory, a recent screening of rice cultivars with respect to endophytic nifH::gus expression revealed significant, cultivar-specific differences in response to Azoarcus sp. strain BH72. Some cultivars supported high levels of root-associated nifH::gus expression, while no significant expression could be detected in others. All of the latter cultivars showed browning of roots upon inoculation, probably due to lignification and accumulation of phenolic substances. This plant reaction, which resembled a plant defense response, required bacterial colonization: a mutant of Azoarcus sp. strain BH72 lacking type IV pili was previously shown to be deficient in effective colonization of rice roots (Dorr et al. 1998). Type IV pili are also known in animal and human pathogens as virulence factors mediating attachment to the host tissue. This mutant did not cause the plant response upon inculation, indicating physical contact of roots and bacteria is necessary to elicit that plant response. Further studies on plant defense genes which might be induced are on the way.
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