A nuclear factor Y interacting protein of the GRAS family is required for nodule organogenesis, infection thread progression, and lateral root growth

Abstract

A C subunit of the heterotrimeric nuclear factor Y (NF-YC1) was shown to play a key role in nodule organogenesis and bacterial infection during the nitrogen fixing symbiosis established between common bean (Phaseolus vulgaris) and Rhizobium etli. To identify other proteins involved in this process, we used the yeast (Saccharomyces cerevisiae) two-hybrid system to screen for NF-YC1-interacting proteins. One of the positive clones encodes a member of the Phytochrome A Signal Transduction1 subfamily of GRAS (for Gibberellic Acid-Insensitive (GAI), Repressor of GAI, and Scarecrow) transcription factors. The protein, named Scarecrow-like13 Involved in Nodulation (SIN1), localizes both to the nucleus and the cytoplasm, but in transgenic Nicotiana benthamiana cells, bimolecular fluorescence complementation suggested that the interaction with NF-YC1 takes place predominantly in the nucleus. SIN1 is expressed in aerial and root tissues, with higher levels in roots and nodules. Posttranscriptional gene silencing of SIN1 using RNA interference (RNAi) showed that the product of this gene is involved in lateral root elongation. However, root cell organization, density of lateral roots, and the length of root hairs were not affected by SIN1 RNAi. In addition, the expression of the RNAi of SIN1 led to a marked reduction in the number and size of nodules formed upon inoculation with R. etli and affected the progression of infection threads toward the nodule primordia. Expression of NF-YA1 and the G2/M transition cell cycle genes Cyclin B and Cell Division Cycle2 was reduced in SIN1 RNAi roots. These data suggest that SIN1 plays a role in lateral root elongation and the establishment of root symbiosis in common bean.

Figure 1.

SIN1 encodes a GRAS transcription factor of the PAT1 subfamily. A, Phylogenetic analysis of the GRAS family of common bean and Arabidopsis based on the classification in subfamilies performed by Bolle (2004) and Pysh et al. (1999). The position of SIN1 (Phvul.003G085000) is indicated by a rectangle. NSP1 and NSP2 from M. truncatula are indicated with arrows. The phylogram was constructed using the neighbor-joining method based on the multiple sequence alignment analysis. The phylogenetic tree was generated using MEGA5 from a ClustalW analysis. Numbers represent bootstrap values obtained from 1,000 trials. B, SCL13 and its homologs from common bean, M. truncatula, and L. japonicus.

Figure 2.

Interaction of NF-YC1 and SIN1. A, Schematic representation of NF-YC1 and deleted versions designed to test the interaction. B, Interaction in yeast using the two-hybrid system. The pGBKT7 plasmid containing the binding domain of the yeast transcription factor Gal4 (BD) was fused to different versions of NF-YC1 and introduced into the Y187 strain by transformation, whereas pGADT7, containing the activation domain of Gal4 (AD), was fused to SIN1 and introduced into the AH109 strain. These strains were mated in the combinations indicated (1 to 8) and selected in synthetic defined media (SD) lacking Leu, Trp (SD-TL) or Leu, Trp, adenine, and His (SD-TLAH). Positive and negative controls are p53 interacting with AgT and LamC, respectively. C, β-Galactosidase activity measured on the diploid yeasts. D, BiFC assay showing the interaction of NF-YC1 and SIN1 in N. benthamiana epidermal cells. The interaction between M. truncatula NSP1 and NSP2 is shown as a positive control. Arrows indicate the fluorescent signal in the nuclei. Bar = 25 μm. E, Subcellular localization of SIN1: the ORF of SIN1 was cloned in pMDC43 for the construction of the GFP-SIN1 fusion and introduced in N. benthamiana leaf cells by agroinfiltration. The same image, obtained with a confocal microscope, is shown under UV (left), white light (middle), and merged (right). Bar = 25 μm. F, Proteins were extracted from leaves agroinfiltrated with GFP-SIN1 or an untransformed strain of Agrobacterium tumefaciens (control), subjected to SDS-PAGE, and analyzed by immunoblot with anti-GFP antibodies, revealing the presence of a single band with the expected size of 88 kD (left). PS, Ponceau stain.

Figure 3.

Relative expression of SIN1 in different organs. A, RT-qPCR analysis of SIN1 accumulation in different organs of common bean. Total RNA was extracted from different organs (leaf, root, and stem) of 7-d-old plants grown under optimal conditions and nodules formed by R. etli SC15 strain at 7 and 14 dpi. B, Levels of SIN1 mRNA in roots after inoculation with R. etli strain SC15 or with yeast-extract mannitol media (control) at 1 or 4 dpi. Expression of the SIN1 gene was measured by RT-qPCR and then normalized with PvEF1α expression values. Data are the media of three technical replicates and are representative of two independent experiments. In B, values are presented relative to the control at 1 dpi. Error bars represent the sd. Expression in roots and 14-dpi nodules were significantly higher than in other tissues in an unpaired two-tailed Student’s t test with P < 0.05. No significant differences were observed between control and inoculated values at 1 or 4 dpi.

Figure 4.

The length of lateral roots is affected in SIN1 RNAi composite plants. A, General cell organization of the root tip of GUS (left) or SIN1 (right) RNAi plants. Cells were observed in a confocal fluorescent microscope with optimal settings for GFP. Images were obtained by z integration of 5-μm sections. Bar = 100 μm. B, The first and second branches of roots emerging from the main hairy root were referred as LR1 and LR2, respectively. Length of the main root, LR1, and LR2 (C) and density of LR1 (D) were measured in GUS- and SIN1 RNAi-transformed roots (black and white bars, respectively). E, Root hair length was also determined in roots inoculated with R. etli SC15 or control (yeast-extracted mannitol). Asterisks in C indicate significant differences in an unpaired two-tailed Student’s t test with P < 0.001 (n > 100). No significant differences were observed in D and E between GUS and SIN1 RNAi values (n > 100). [See online article for color version of this figure.]

Figure 5.

SIN1 RNAi plants developed less and smaller nodules than controls. Nodulation phenotype of SIN1 (A) and GUS RNAi (B) roots at 7 dpi with R. etli SC15. The number (C) and size (D) of nodules were recorded in both types of plants at different times after inoculation. Asterisks in D indicate significant differences in an unpaired two-tailed Student’s t test with P < 0.001 (n > 120). Occupancy of nodules was examined 5 dpi with a strain of R. etli that expresses the DsRed protein by fluorescent microscopy in SIN1 (E and F) and GUS RNAi roots (G and H) under UV (E and G) or visible light (F and H). Longitudinal sections of nodules formed in GUS (I) or SIN1 (J) RNAi roots were observed at 21 dpi with R. etli strain SC15. Bars = 1 cm (A and B), 500 μm (E–H), and 300 μm (I and J).

Figure 6.

Effect of SIN1 RNAi on infection events. The density of ITs (number of IT per root centimeter) formed in GUS and SIN1 RNAi composite plants was quantified at 4 and 8 dpi with a R. etli strain that expresses the DsRed protein. There were no significant differences between GUS and SIN1 RNAi at 4 or 8 dpi in an unpaired two-tailed Student’s t test with P < 0.05 (A). ITs formed were classified as events that reach the cortex (white bars), end in the epidermis (gray bars), or end in the root hair (RH; black bars) and expressed as percentage of the total. The number of ITs that end in RH or reach the cortex were significantly higher in SIN1 than in GUS RNAi plants in an unpaired two-tailed Student’s t test with P < 0.05, whereas ITs that end in epidermis did not show significant variations (B). Approximately 60 infection events were recorded for GUS and SIN1 RNAi at 4 or 8 dpi.

Figure 7.

Effect of SIN1 RNAi on the expression of early nodulins (A) and cell cycle genes (B). GUS (black bars) or SIN1 RNAi (white bars) roots were inoculated with the R. etli strain SC15 and tissue was collected 6 or 24 hpi. Controls were treated with yeast-extract mannitol for 24 h. Expression of indicated genes was measured by RT-qPCR, normalized with PvEF1α expression values, and presented relative to the values of GUS RNAi controls. Data are the media of three technical replicates, and two other independent experiments are shown in Supplemental Figure S7. Error bars represent the sd. Asterisks indicate that expression values in SIN1 RNAi are significantly different from those in GUS RNAi roots at the same time point, in an unpaired two-tailed Student’s t test with P < 0.01.