Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae

Abstract

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Determination of symbiotic nodule occupancy in the model Vicia tetrasperma using a fluorescence scanner

“…The excitation and emission maxima of cycle3 GFP are 395 and 508 nm, respectively, thus corresponding to the published wild-type GFP (Cubitt et al, 1995). The in vivo spectrum did not change after cycle3 transfer from E. coli into the Rhizobium background despite the sharp decrease in fluorescence yield (Chovanec et al, 2008). The bacteria were grown on yeast extract-mannitol (YM) agar with kanamycin at 50 mg mL 21 as a transposon-stabilizing antibiotic (Chovanec et al, 2008) for 4 d at 25 8C.…”

“…Rhizobium leguminosarum 128C30 (EMD Crop Bioscence, formerly Nitragin, Milwaukee, WI, USA) is a wild-type strain containing symbiotic megaplasmid pSym128C30 of 212 MDa (¼ 326 kbp; Leyva et al, 1987). Its fluorescent variant strain, 128C30(1819/9), was prepared by in vivo marking with a minitransposon Tn1819 upon conjugative transfer on a suicidal plasmid pFAJ1819 from Escherichia coli S17-1lpir according to Chovanec et al (2008). The plasmid pFAJ1819 is a derivative of pUT (Herrero et al, 1990) prepared by Xi et al (1999) while Tn1819 (Xi et al, 1999) is a mini-Tn5 derivative that contains two copies of the optimized GFP gene put under control of the constitutive promoter of nptII.…”

“…The in vivo spectrum did not change after cycle3 transfer from E. coli into the Rhizobium background despite the sharp decrease in fluorescence yield (Chovanec et al, 2008). The bacteria were grown on yeast extract-mannitol (YM) agar with kanamycin at 50 mg mL 21 as a transposon-stabilizing antibiotic (Chovanec et al, 2008) for 4 d at 25 8C. The inoculum was prepared by scraping colonies off the surface of the YM agar plates, resuspending bacteria in distilled water and adjusting the titre by direct counting.…”