In Situ Identification of Plant-Invasive Bacteria with MALDI-TOF Mass Spectrometry

Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues

Nodulation in the legume biofuel feedstock tree Pongamia pinnata

The legume tree Pongamia pinnata (also called Millettia pinnata) is gaining importance as a biofuel feedstock tree because of the abundant annual production of oil-rich seeds, adaptation to a wide range of geoclimatic conditions and significant resistance to abiotic stress, such as water-deficit, salinity and acidity of soils. The major defining benefit of using pongamia as a biofuel feedstock is that it is a legume, enabling biological nitrogen fixation through symbiosis with soil bacteria, collectively called rhizobia, which results in root nodulation. Here, we report preliminary data, (i) indicating the range of rhizobia that can form nodules on pongamia, (ii) demonstrating the measurement of nitrogen fixation activity of pongamia nodules via the classical acetylene reduction assay, (iii) illustrating nodule morphology and development and (iv) demonstrating aspects of nodule regulation by external nitrate as well as internal autoregulation of nodulation. We note that in pongamia most nodulation-related characteristics are similar to those found in other annual crop legumes such as soybean

A novel method based on combination of semi-in vitro and in vivo conditions in Agrobacterium rhizogenes-mediated hairy root transformation of Glycine species

Despite numerous advantages of the many tissue culture-independent hairy root transformation protocols, the process is often compromised in the initial in vitro culture stage where inability to maintain high humidity and the delivery of nourishing culture medium decrease cellular morphogenesis and organ formation efficiency. Ultimately, this influences the effective transfer of produced plantlets during transfer from in vitro to in vivo conditions, where low survival rates occur during the acclimation period. We have developed an intermediate protocol for Agrobacterium rhizogenes transformation in Glycine species by combining a two-step in vitro and in vivo process that greatly enhances the efficiency of hairy root formation and which simplifies the maintenance of the transformed roots. In this protocol, cotyledonary nodes of Glycine max and Glycine canescens seedlings were infected by A. rhizogenes K599 carrying a reporter gene construct constitutively expressing green fluorescent protein (GFP). Glass containers containing sand and nutrient solution were employed to provide a moist clean microenvironment for the generation of hairy roots from inoculated seedlings. Transgenic roots were then noninvasively identified from nontransgenic roots based on the detection of GFP. Main roots and nontransgenic roots were removed leaving transgenic hairy roots to support seedling development, all within 1 mo of beginning the experiment. Overall, this protocol increased the transformation efficiency by more than twofold over traditional methods. Approximately 88% and 100% of infected plants developed hairy roots from G. max and G. canescens, respectively. On average, each infected plant produced 10.9 transformed hairy roots in G. max and 13–20 in G. canescens. Introduction of this simple protocol is a significant advance that eliminates the long and genotype-dependent tissue culture procedure while taking advantage of its optimum in vitro qualities to enhance the micropropagation rate. This research will support the increasing use of transient transgenic hairy roots for the study of plant root biology and symbiotic interactions with Rhizobium spp.Manijeh Mohammadi-Dehcheshmeh, Esmaeil Ebrahimie, Stephen D. Tyerman, Brent N. Kaise