Local and Systemic Regulation of Plant Root System Architecture and Symbiotic Nodulation by a Receptor-Like Kinase

In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.     

Developmental analysis of a Medicago truncatula smooth leaf margin1 mutant reveals context-dependent effects on compound leaf development

Compound leaf development requires highly regulated cell proliferation, differentiation, and expansion patterns. We identified loss-of-function alleles at the SMOOTH LEAF MARGIN1 (SLM1) locus in Medicago truncatula, a model legume species with trifoliate adult leaves. SLM1 encodes an auxin efflux carrier protein and is the ortholog of Arabidopsis thaliana PIN-FORMED1 (PIN1). Auxin distribution is impaired in the slm1 mutant, resulting in pleiotropic phenotypes in different organs. The most striking change in slm1 is the increase in the number of terminal leaflets and a simultaneous reduction in the number of lateral leaflets, accompanied by reduced expression of SINGLE LEAFLET1 (SGL1), an ortholog of LEAFY. Characterization of the mutant indicates that distinct developmental domains exist in the formation of terminal and lateral leaflets. In contrast with the pinnate compound leaves in the wild type, the slm1 sgl1 double mutant shows nonpeltately palmate leaves, suggesting that the terminal leaflet primordium in M. truncatula has a unique developmental mechanism. Further investigations on the development of leaf serrations reveal different ontogenies between distal serration and marginal serration formation as well as between serration and leaflet formation. These data suggest that regulation of the elaboration of compound leaves and serrations is context dependent and tightly correlated with the auxin/SLM1 module in M. truncatula.     

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against Pseudomonas syringae Pathogens

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

Flexible functional interactions between G‐protein subunits contribute to the specificity of plant responses

Plants being sessile integrate information from a variety of endogenous and external cues simultaneously to optimize growth and development. This necessitates the signaling networks in plants to be highly dynamic and flexible. One such network involves heterotrimeric G‐proteins comprised of Gα, Gβ, and Gγ subunits, which influence many aspects of growth, development, and stress response pathways. In plants such as Arabidopsis, a relatively simple repertoire of G‐proteins comprised of one canonical and three extra‐large Gα, one Gβ and three Gγ subunits exists. Because the Gβ and Gγ proteins form obligate dimers, the phenotypes of plants lacking the sole Gβ or all Gγ genes are similar, as expected. However, Gα proteins can exist either as monomers or in a complex with Gβγ, and the details of combinatorial genetic and physiological interactions of different Gα proteins with the sole Gβ remain unexplored. To evaluate such flexible, signal‐dependent interactions and their contribution toward eliciting a specific response, we have generated Arabidopsis mutants lacking specific combinations of Gα and Gβ genes, performed extensive phenotypic analysis, and evaluated the results in the context of subunit usage and interaction specificity. Our data show that multiple mechanistic modes, and in some cases complex epistatic relationships, exist depending on the signal‐dependent interactions between the Gα and Gβ proteins. This suggests that, despite their limited numbers, the inherent flexibility of plant G‐protein networks provides for the adaptability needed to survive under continuously changing environments.     

Inhibition of Helicobacter pylori growth and its cytotoxicity by 2-hydroxy 4-methoxy benzaldehyde of Decalepis hamiltonii (Wight & Arn); a new functional attribute

Helicobacter pylori mediated gastric ulcer and cancers are common global problems since it was found to colonize in ∼50% of gastric ulcer/cancer patients. Decalepis hamiltonii, (Asclepiadaceae family) extracts have been depicted with medicinal properties supporting the traditional knowledge of health beneficial attributes of D. hamiltonii. Previously we have shown that both aqueous as well as methanol extracts of D. hamiltonii containing abundant phenolics with predominant levels (20-40% of total phenolics) of 2-hydroxy-4-methoxy benzaldehyde (HMBA). Despite higher levels, HMBA contributed very little to the antioxidant activity (<10%) when compared to other phenolic compounds in the extract. In the current study we attempted to explore antimicrobial property, particularly anti-H. pylori activity, since traditional users document D. hamiltonii as a fighter of microbial infections. HMBA was isolated from the roots of D. hamiltonii by hydrodistillation and cold crystallization method; identified by HPLC and characterized using ESI-MS and confirmed by NMR studies as a compound of molecular mass 152 Da. Isolated HMBA was found to inhibit the growth of H. pylori, a potential ulcerogen in a dose dependent manner with MIC of ∼39 μg/mL as apposed to that of amoxicillin (MIC - 26 μg/mL) for which H. pylori is susceptible. Results were further substantiated by the lysis of H. pylori by electron microscopy and electrophoretic studies. Studies on the mechanism of action indicated the counteracting effect of vacuolating toxin (VacA) of H. pylori which otherwise would lead to host cell cytotoxicity. Further the increased binding ability of HMBA to DNA and protein offered an impact on DNA protectivity and bioavailability. Results for the first time provide a direct evidence for anti-microbial attribute of HMBA. Insignificant antioxidant attribute of HMBA also reveals the anti-H. pylori activity via mechanisms other than antioxidative routes.     

Brain development in the yellow fever mosquito Aedes aegypti: a comparative immunocytochemical analysis using cross-reacting antibodies from Drosophila melanogaster

Considerable effort has been directed towards understanding the organization and function of peripheral and central nervous system of disease vector mosquitoes such as Aedes aegypti. To date, all of these investigations have been carried out on adults but none of the studies addressed the development of the nervous system during the larval and pupal stages in mosquitoes. Here, we first screen a set of 30 antibodies, which have been used to study brain development in Drosophila, and identify 13 of them cross-reacting and labeling epitopes in the developing brain of Aedes. We then use the identified antibodies in immunolabeling studies to characterize general neuroanatomical features of the developing brain and compare them with the well-studied model system, Drosophila melanogaster, in larval, pupal, and adult stages. Furthermore, we use immunolabeling to document the development of specific components of the Aedes brain, namely the optic lobes, the subesophageal neuropil, and serotonergic system of the subesophageal neuropil in more detail. Our study reveals prominent differences in the developing brain in the larval stage as compared to the pupal (and adult) stage of Aedes. The results also uncover interesting similarities and marked differences in brain development of Aedes as compared to Drosophila. Taken together, this investigation forms the basis for future cellular and molecular investigations of brain development in this important disease vector.