Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs

Abstract

SUMMARYRoot hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca 2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.

Rapid Phosphoproteomic and Transcriptomic Changes in the Rhizobia-legume Symbiosis

“…Small G proteins (guanine nucleotide-binding proteins) are known to be involved in the regulation of the phosphoinositide pathway, vesicle trafficking, root hair growth, and the early steps of legume nodulation (42,108,114,115). The role of heterotrimeric G proteins in early symbiotic signaling was also proposed.…”

“…The LYR1-RNAi fragment was cloned into pK7GWIWG2-R hairpin RNAi destination vector carrying DsRed1 as visible reporter by LR-recombination. Medicago Jemalong A17 seedlings were transformed with LYR1-RNAi cassette by Agrobacterium rhizogenes (strain MSU440) -mediated hairy root transformation (42). Transgenic roots expressing LYR1-RNAi cassette were identified by DsRED1 expression (supplemental Fig.…”

“…We took a candidate reverse genetics approach to explore the possibility that LYR1 could mediate the transcriptional responses in nfp upon NF treatment. Using RNA interference, we silenced LYR1 in wild-type and nfp plants as described previously (42,57). Transgenic roots containing the RNA interference construct were analyzed for transcriptional changes one hour post-NF treatment by qRT-PCR.…”

The Recent Evolution of a Symbiotic Ion Channel in the Legume Family Altered Ion Conductance and Improved Functionality in Calcium Signaling

“…A 451-bp fragment at the 59 coding region of Mt-CASTOR was amplified using the primers P 6 and P 7 and cloned into a modified hairpin RNAiexpressing binary vector, pK7GWIWG2(II)-R, containing the constitutively expressed fluorescent marker DsRED1 (Riely et al, 2011). As a positive control, we performed RNAi-based gene knockdown of DMI1.…”

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…Various regulators that modulate the shuttling between inactive and active forms of ROP GTPases have been identified, including guanine nucleotide exchange factors (RopGEFs) that stimulate GDP-to-GTP exchange to activate ROP GTPases, ROP GTPase-activating proteins (RopGAPs) that accelerate GTP hydrolysis, and guanine nucleotide dissociation inhibitors (RhoGDIs) that inhibit GDP release, thus shifting ROP GTPases toward the inactive state. Some of these regulators have been shown to modulate ROP GTPase activity in pollen tubes and root hairs (Carol et al, 2005;Gu et al, 2006;Zhang and McCormick, 2007;Hwang et al, 2008Hwang et al, , 2010Riely et al, 2011).…”