Mark Kinkema

Nuclear localization of NPR1 is required for activation of PR gene expression.

Systemic acquired resistance (SAR) is a broad-spectrum resistance in plants that involves the upregulation of a battery of pathogenesis-related (PR) genes. NPR1 is a key regulator in the signal transduction pathway that leads to SAR. Mutations in NPR1 result in a failure to induce PR genes in systemic tissues and a heightened susceptibility to pathogen infection, whereas overexpression of the NPR1 protein leads to increased induction of the PR genes and enhanced disease resistance. We analyzed the subcellular localization of NPR1 to gain insight into the mechanism by which this protein regulates SAR. An NPR1–green fluorescent protein fusion protein, which functions the same as the endogenous NPR1 protein, was shown to accumulate in the nucleus in response to activators of SAR. To control the nuclear transport of NPR1, we made a fusion of NPR1 with the glucocorticoid receptor hormone binding domain. Using this steroid-inducible system, we clearly demonstrate that nuclear localization of NPR1 is essential for its activity in inducing PR genes.

Agrobacterium rhizogenes-mediated transformation of soybean to study root biology.

This protocol is used to induce transgenic roots on soybean to study the function of genes required in biological processes of the root. Young seedlings with unfolded cotyledons are infected at the cotyledonary node and/or hypocotyl with Agrobacterium rhizogenes carrying the gene construct to be tested and the infection sites are kept in an environment of high humidity. When the emerged hairy roots can support the plants, the main roots are removed and the transgenic roots can be tested. Using this method, almost 100% of the infected plants form hairy roots within 1 month from the start of the experiments.

Legume nodulation: successful symbiosis through short- and long-distance signalling

Nodulation in legumes provides a major conduit of available nitrogen into the biosphere. The development of nitrogen-fixing nodules results from a symbiotic interaction between soil bacteria, commonly called rhizobia, and legume plants. Molecular genetic analysis in both model and agriculturally important legume species has resulted in the identification of a variety of genes that are essential for the establishment, maintenance and regulation of this symbiosis. Autoregulation of nodulation (AON) is a major internal process by which nodule numbers are controlled through prior nodulation events. Characterisation of AON-deficient mutants has revealed a novel systemic signal transduction pathway controlled by a receptor-like kinase. This review reports our present level of understanding on the short- and long-distance signalling networks controlling early nodulation events and AON.

Investigation of Downstream Signals of the Soybean Autoregulation of Nodulation Receptor Kinase GmNARK

The Glycine max nodule autoregulation receptor kinase (GmNARK) plays a central role in the systemic signal transduction pathway controlling nodulation in soybean. We used transcriptional profiling to identify potential downstream signals of this receptor kinase. These studies revealed that GmNARK-mediated signaling controls the expression of genes involved in the jasmonic acid (JA) pathway. Genes encoding the key enzymes controlling JA biosynthesis as well as JA-response genes were regulated systemically but not locally by root inoculation with Bradyrhizobium japonicum. This systemic regulation was abolished in Gmnark mutant plants, indicating that their expression was specifically controlled by signaling events associated with this receptor kinase. Foliar application of a JA biosynthesis inhibitor significantly reduced nodulation specifically in supernodulating mutant plants. These results indicate that the receptor-mediated regulation of JA signaling plays an important role in the AON signal transduction pathway. A second class of genes was identified that were controlled by GmNARK in a rhizobia-independent manner. These candidates provide insight on additional, nonsymbiotic signaling pathways that are likely regulated by GmNARK, such as those involved in root growth and defense. The discovery of downstream components of the GmNARK receptor kinase advances our understanding of the systemic control of nodule development and its association with other signaling networks.

Molecular analysis of lipoxygenases associated with nodule development in soybean.

We utilized transcriptional profiling to identify genes associated with nodule development in soybean. Many of the candidate genes were predicted to be involved in processes such as defense, metabolism, transcriptional regulation, oxidation, or iron storage. Here, we describe the detailed characterization of one specific class of genes that encode the enzyme lipoxygenase (LOX). The LOX9 and LOX10 genes identified by microarray analysis represent novel soybean LOXs expressed in developing nodules. LOX expression during nodulation was relatively complex, with at least eight different LOX genes expressed in soybean nodules. Histochemical analyses utilizing LOX9 promoter::beta-glucuronidase (GUS) fusion constructs in transgenic soybean hairy roots suggest that this gene is involved in the growth and development of specific cells within the root and nodules. In soybean roots, LOX9 was expressed specifically in the developing phloem. In nodules, the expression of LOX9 was correlated with the development of cells in the vasculature and lenticels. The use of RNAi in transgenic hairy roots reduced LOX expression by approximately 95%. Despite this significant reduction in LOX expression, there was no detectable effect on the development of roots or nodules. Our findings are discussed with respect to the potential function of LOXs in nodulation.

Agglutinins from marine macroalgae of the southeastern United States

Protein extracts from 22 species of marine macroalgae from Florida and North Carolina were compared for their abilities to agglutinate sheep and rabbit erythrocytes. Protein extracts from 21 algal species agglutinated rabbit erythrocytes compared to 19 for sheep erythrocytes. However, agglutination by brown algal extracts was variable. The agglutination produced by protein extracts from Dictyota dichotoma could be blocked by addition of polyvinylpyrrolidone. Protein extracts from North Carolina macroalgae were also tested against five bacterial species. Three of these agglutinated bacterial cells. Ulva curvata and Bryopsis plumosa agglutinated all five species. Protein extracts from five species of Florida algae were tested for their effects on mitogenesis in mouse splenocytes and human lymphocytes. Gracilaria tikvahiae HBOI Strain G-5, Ulva rigida and Gracilaria verrucosa HBOI Strain G-16S stimulated mitogenesis in mouse splenocytes, while Gracilaria tikvahiae HBOI Strain G-16stimulated mitogenesis in human lymphocytes.

Improved molecular tools for sugar cane biotechnology

Abstract Sugar cane is a major source of food and fuel worldwide. Biotechnology has the potential to improve economically-important traits in sugar cane as well as diversify sugar cane beyond traditional applications such as sucrose production. High levels of transgene expression are key to the success of improving crops through biotechnology. Here we describe new molecular tools that both expand and improve gene expression capabilities in sugar cane. We have identified promoters that can be used to drive high levels of gene expression in the leaf and stem of transgenic sugar cane. One of these promoters, derived from the Cestrum yellow leaf curling virus, drives levels of constitutive transgene expression that are significantly higher than those achieved by the historical benchmark maize polyubiquitin-1 (Zm-Ubi1) promoter. A second promoter, the maize phosphonenolpyruvate carboxylate promoter, was found to be a strong, leaf-preferred promoter that enables levels of expression comparable to Zm-Ubi1 in this organ. Transgene expression was increased approximately 50-fold by gene modification, which included optimising the codon usage of the coding sequence to better suit sugar cane. We also describe a novel dual transcriptional enhancer that increased gene expression from different promoters, boosting expression from Zm-Ubi1 over eightfold. These molecular tools will be extremely valuable for the improvement of sugar cane through biotechnology.

Nodulation Control in Legumes

Nodulation and concomitant symbiotic nitrogen fixation are critical for the productivity of the legume, yielding food, feed and fuel. The nodule number in legumes is regulated by numerous factors including the number and efficiency of the interacting Rhizobium bacteria and abiotic stresses as well as endogenous processes involving phytohormones, nodulation reception systems and autoregulation of nodulation (AON; Kinkema et al., 2006). The original discovery of the AON-controlling LRR receptor kinases, GmNARK/ LjHAR1/MtSUNN, which is active in leaf tissue of several legu-mes, now has led to an analysis of the mechanism underlying the signal transduction.

Functional Genomics of the Regulation of Nodule Number in Legumes

Peter M. Gresshoff, Gustavo Gualtieri, Titeki Laniya, Arief Indrasumunar, Akira Miyahara, Sureeporn Nontachaiyapoom, Tim Wells, Bandana Biswas, Pick Kuen Chan, Paul Scott, M. Kinkema, M. Djordjevic, Dana Hoffmann, Lisette Pregelj, Diana M. Buzas, Dong Xi Li, Artem Men, Qunyi Jiang, Cheol-Ho Hwang and Bernard J. Carroll ARC Centre of Excellence for Integrative Legume Research; School of Life Sciences, and School of Molecular and Microbial Sciences and LAFS, The University of Queensland, St. Lucia, Brisbane QLD 4072, AGRF; Genome Interaction Group, RSBS, ANU, Canberra, ACT, Australia.