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The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation - PubMed

  • ️Wed Dec 23 2229

doi: 10.1105/tpc.114.135210. Epub 2015 Mar 20.

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The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation

Ming-Juan Lei et al. Plant Cell. 2015 Mar.

Abstract

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.

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Figures

Figure 1.
Figure 1.

Root Hair Phenotypes of Transformed M. truncatula Roots Overexpressing ROP10 and ROP10CA or ROP10DN. (A) and (B) Roots harboring the empty vector showed normal tip growth of root hairs. (C) to (F) Roots overexpressing ROP10 produced root hairs with weakly ([C] and [D]) to strongly ([E] and [F]) depolarized growth. (G) to (J) Roots overexpressing ROP10CA produced short swollen root hairs ([G] and [H]) as well as completely ballooning root hairs ([I] and [J]). (K) and (L) Roots overexpressing ROP10DN produced short root hairs with almost normal tip growth. About 120 composite plants were obtained for each construct in each experiment, and over 40% of composite plants generated transformed roots. The hairy root transformation experiment for each construct was repeated at least five times. Bars in (A), (C), (E), (G), (I), and (K) = 500 µm; bars in in (B), (D), (F), (H), (J), and (L) = 100 µm. (M) Quantitative analysis of the average length of root hairs in transformed roots overexpressing the indicated proteins. Ten root hair cells were measured per transformed root, and 15 transformed roots were scored. Error bars indicate

se

. Statistical significance (*P < 0.05, **P < 0.01) was evaluated by Student’s t test. (N) Quantitative RT-PCR of ROP10 mRNA levels in transformed roots overexpressing ROP10 that showed weakly depolarized root hairs (ROP10-weak) and strongly depolarized root hairs (ROP10-strong). Statistical significance (**P < 0.01) was evaluated by Student’s t test. Error bars indicate

se

. Data presented are representative of three independent experiments.

Figure 2.
Figure 2.

Subcellular Localization of ROP10:GFP and Mutant Forms in N. benthamiana Leaf Epidermal Cells. (A) ROP10:GFP, ROP10CA:GFP, and ROP10DN:GFP were localized exclusively at the PM, whereas free GFP was distributed in the PM, cytoplasm, and nuclei. Bars = 50 μm. (B) Inhibition of S-acylation of ROP10:GFP, ROP10CA:GFP, and ROP10DN:GFP by a treatment with 2-bromopalmitate resulted in release of the expressed proteins from the PM. Fluorescence is observed in the cytoplasm and nuclei (arrowheads). Bars = 50 μm. (C) ROP10 proteins with mutations in the GC-CG box (ROP10C203S:GFP, ROP10C197S:GFP, and ROP10C197+203S:GFP) and ROP10CA mutant with mutations (ROP10CAC197+203S:GFP) showed reduced association with the PM, and fluorescence is observed in the cytoplasm and nuclei (arrowheads). Bars = 50 μm. (D) Immunoblotting using GFP monoclonal antibody. Soluble (S) and insoluble membrane (P) fractions were separated from N. benthamiana leaf cells overexpressing different forms of ROP10 proteins. ROP10, ROP10CA, and ROP10DN were detected in the membrane fraction, and free GFP was detected in both the soluble and membrane fractions.

Figure 3.
Figure 3.

ROP10:GFP Is Localized at the Apical PM of Root Hairs and the PM of Outgrowths and Curling in Response to NF Treatment and Rhizobial Infection. (A) and (B) Subcellular localization of free GFP in root epidermal cells (A) and root hairs (B) of control roots expressing GFP alone. (C) ROP10:GFP is localized at the PM of root epidermal cells close to the root tip. (D) ROP10:GFP is localized at the PM of root hair bulges, which produce elongating root hairs by tip growth. (E) ROP10:GFP is localized at the PM in the apex of elongating root hairs close to the root tip. (F) ROP10:GFP is localized at the PM throughout the ballooning root hairs. (G) Following treatment with 1 nM NodSm-IV(C16:2, S) for 24 h, ROP10:GFP is localized at the PM of outgrowths (arrowheads) of a swollen root hair. (H) ROP10:GFP is localized at the PM of outgrowths (arrowheads) of a swollen root hair 7 d after inoculation with S. meliloti. (I) ROP10:GFP is visible in the curling (green) of the root hair 5 d after inoculation with mCherry-expressing S. meliloti (red; arrowhead). Bars = 100 μm.

Figure 4.
Figure 4.

Overexpression of Wild-Type ROP10 and ROP10CA Affects Rhizobial Infection and Nodulation of M. truncatula. (A) to (D) Control roots transformed with the empty vector showed a normal infection process of root hairs. Rhizobia are present in the infection pocket of a curled root hair tip (A), in the infection thread that extends within a curled root hair (B), in infection threads that ramify into a fine network in the nodule primordium (C), and in a developing nodule (D). (E) to (L) Roots overexpressing ROP10 showed an aberrant infection process in swollen root hairs. Microcolonies of bacteria were often absent in swollen root hairs (E). Frequently, more than one outgrowth in a single root hair were observed ([F] to [H]) and infection threads aborted due to the formation of a sac-like structure (arrowhead) (I). Occasionally, a new infection thread was initiated from the sac-like structure and reached the root cortex (J). Double infection threads were also observed in a single root hair ([K] and [L]). Rhizobia were detected by β-galactosidase staining with 5-bromo-4-chloro-3-indolyl β-

d

-galactopyranoside. Photographs were taken 7 d after inoculation with S. meliloti. Bars = 100 μm. (M) Overexpression of ROP10 and ROP10CA led to a significant reduction of root hair infection. Infection events include observed infection foci and infection threads. The infection events were scored at 7 d after inoculation. (N) Overexpression of ROP10 and ROP10CA resulted in reduced nodulation. The number of nodules on transgenic roots per plant was scored at 14 d after inoculation. Statistical significance (**P < 0.01) was evaluated by Student’s t test. Error bars indicate

se

. Data presented are representative of three independent experiments.

Figure 5.
Figure 5.

Interactions between Different Forms of ROP10 and the NF Receptor NFP of M. truncatula. (A) GAL4-based Y2H assays. ROP10 and mutant forms (ROP10CA and ROP10DN) were fused with Gal4 DNA BD in pGBKT7 as baits, and NFP full length (FL), the intracellular kinase domain of NFP (PK), LYK3 PK, and LYK3 FL were fused with the Gal4 AD in pGADT7 as preys. Yeast cells cotransformed with bait and prey constructs were selected on SD medium lacking His, Leu, and Trp supplemented with X-α-Gal (SD/-3/X-α-Gal) and more stringent SD medium lacking His, Ade, Leu, and Trp supplemented with X-α-Gal (SD/-4/X-α-Gal) for 5 d. The results indicate an interaction between NFP PK and ROP10 or ROP10CA. The interaction between mammalian p53 and SV40 served as a positive control, whereas the coexpression of lamin (Lam) and SV40 served as a negative control. (B) The strength of interaction was quantified by assaying β-galactosidase activities in yeast colonies with chlorophenol red-β-

d

-galactopyranoside as substrate. Error bars indicate

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. Data presented are representative of at least three independent experiments. (C) Colocalization of ROP10 and NFP at the PM in N. benthamiana leaf epidermal cells. Bars = 50 µm. (D) BiFC analysis to test interactions between ROP10 or mutant forms and NFP PK in N. benthamiana leaf epidermal cells. Bars = 50 µm.

Figure 6.
Figure 6.

NF-Induced ENOD11 Expression Is Enhanced in Transformed M. truncatula Roots Overexpressing ROP10 or ROP10CA. (A) Transgenic roots overexpressing ROP10 or ROP10CA were generated by A. rhizogenes-mediated transformation of M. truncatula line 416 harboring an ENOD11pro:GUS fusion. Roots were stained with 5-bromo-4-chloro-3-indolyl-β-

d

-glucuronic acid after treatment for 12 h with 1 nM NodSm-IV(C16:2, S). Control roots transformed with the empty vector had a typical NF-induced ENOD11 expression pattern, whereas roots overexpressing ROP10 or ROP10CA showed more extended and stronger GUS staining. Bars = 5 mm. (B) and (C) Fluorometric analysis of GUS activity (B) and qRT-PCR analysis of ENOD11 expression (C) in transformed roots overexpressing ROP10 or ROP10CA 12 h after treatment with 1 nM NodSm-IV(C16:2, S). Asterisks indicate significant increases relative to the empty vector control (P < 0.05) as evaluated using Student’s t test. Error bars indicate

se

. Data presented are representative of three independent experiments.

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