A small GTPase of the Rab family is required for root hair formation and preinfection stages of the common bean-Rhizobium symbiotic association

Abstract

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.

Figure 1.

RHS24-03 Encodes a Protein Closely Related to Members of the RabA2 Subfamily. A phylogenetic tree of Arabidopsis Rab proteins and RHS24-03 (arrow) was generated using MEGA4 from a ClustalW alignment (available as Supplemental Data Set 1 online). Numbers represent bootstrap values obtained from 1000 trials. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary distances are displayed in units of the number of amino acid substitutions per site.

Figure 2.

mRNA of RabA2 Accumulates in Rhizobia-Inoculated Roots and Root Hairs. Total RNA was extracted from root hairs or the whole root from plants inoculated with yeast extract mannitol medium (YEM) or R. etli strain SC15 or 55N1 at 24 HAI. Levels of RabA2 were measured by qRT-PCR and normalized to elongation factor 1α (EF1α) values. Expression was then normalized to values obtained from roots of plants inoculated with YEM. Means ± sd of three technical replicates are presented. Results are representative of three independent experiments.

Figure 3.

RABA2 Localizes to Vesicles inside of Root Hairs. Roots from common bean plants expressing soluble GFP (A) or the GFP-RABA2 fusion ([B] and [C]) were observed by confocal microscopy. Time-lapse images of a root hair expressing GFP-RABA2 were collected every 4 s. Individual frames presented in (B) are part of Supplemental Movie 1 online. A root hair tip from a plant 24 HAI with R. etli is shown in (C). Bars = 20 μm.

Figure 4.

Temporal Pattern of Silencing of RabA2 after A. rhizogenes Transformation. RNA was extracted from globular callus (time 0) and hairy roots at different times after emergence from the callus. Levels of RabA2 were measured by qRT-PCR in GUS and RabA2 RNAi transgenic tissue and normalized to EF1α values. Inoculation with R. etli was performed on a group of plants at day 18 and samples collected daily until 3 d after infection (dashed lines). Means ± sd of three technical replicates are presented.

Figure 5.

Effect of RabA2 Silencing and Overexpression on Root Hairs. RabA2 RNAi (A) or GUS RNAi (B) root hairs were observed under the microscope. Root hairs were counted (C) and measured (D) from at least five different composite plants. Mean values were compared with GUS RNAi according to a Student's t test (*, P < 0.05; n > 500). Bars = 100 μm.

Figure 6.

RNAi Posttranscriptional Silencing and Overexpression of RabA2 in Transformed Hairy Roots. Hairy roots of composite plants grown on solid Fahraeus mineral medium were inoculated with R. etli strain SC15 or 55N1. At 6 d after inoculation, several roots harvested from the same plant were used for RNA extraction. Relative transcript levels of RabA2 (top panel) and RabA1 (bottom panel) in RabA2 RNAi plants were compared with those from plants transformed with the GUS RNAi construct or inoculated with the untransformed A. rhizogenes strain K599 (A). Relative mRNA expression levels of RabA2 in hairy roots overexpressing RabA2 under the control of the CaMV35S promoter (35S:RabA2) were compared with plants transformed with control vector (35S:GFP/GUS) or with the untransformed A. rhizogenes (B). Roots from plants #6 and #7 carrying the 35S:RabA2 construct showed levels of RabA2 similar to 35S:GFP/GUS plants. Transcripts levels were measured by qRT-PCR and normalized to EF1α values. Means ± sd of three technical replicates are presented.

Figure 7.

Effect of RabA2 RNAi Silencing on the Early Symbiotic Responses to Rhizobial Infection. (A) Attachment of R. etli expressing GFP to the surface of root hairs: GUS RNAi transformed roots inoculated with YEM medium (left) or inoculated with GFP-labeled R. etli strain 55N1 at 24 HAI (middle) and RabA2 RNAi transformed roots inoculated with the same strain at 24 HAI (right). Attachment can be visualized as green dots on the surface of root hairs, as indicated by the arrows. Bars =100 μm. (B) Root hair curling observed 24 HAI with R. etli in GUS RNAi (middle) compared with plants treated with YEM (left) or RabA2 RNAi roots (right). Arrows point to curled root hairs. Bars = 50 μm. (C) ITs formed at 48 HAI on GUS RNAi plants (left), showing two ITs in detail. Root hair deformation was absent in RabA2 RNAi roots (right). Bars = 50 μm. Digital photographs shown in (A) were obtained under UV light with the appropriate filter for GFP; images in (B) were obtained with visible light. Epifluorescence and visible light were merged in (C).

Figure 8.

Early Nodulin Expression in RabA2-Silenced Plants. Composite plants carrying the GUS RNAi (black bars) or the RabA2 RNAi construct (white bars) were inoculated with R. etli strain SC15 or with YEM (control). For rhizobia-inoculated plants, root tissue was harvested at the indicated times. Roots from plants inoculated with YEM were harvested at 6, 12, and 24 HAI and then pooled. Total RNA was extracted and transcript levels measured by qRT-PCR. Expression values were normalized to EF1α values. Means ± sd of three technical replicates are presented. Results are representative of three independent biological samples. (A) RabA2 and RabA1 mRNA levels were determined in YEM-inoculated samples. (B) Transcript levels of ENOD40, the C subunit of CCAAT binding factor and ERN were determined in RNA samples collected at each time point after inoculation and presented as relative to the values obtained from the YEM-inoculated GUS RNAi sample.