Genetic diversity of <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> isolates from certain agro-climatic regions of India by using RAPD markers

Genetic diversity analysis of Macrophomina phaseolina isolates obtained from different host range and diverse geographical locations in India was carried out using RAPD fingerprinting. Of the thirteen 10-mer random primers used, primer OPB-08 gave the maximum polymorphism and the UPGMA clustering could separate 50 isolates in to ten groups at more than 65% similarity level. The ten clusters correlated well with the geographical locations with exceptions for isolates obtained from Eastern and Western Ghats. There was a segregation of isolates from these two geographical locations in to two clusters thus, distributing 10 genotypes in to eight geographical locations. All the isolates M. phaseolina irrespective of their host and geographical origin, exhibited two representative monomorphic bands at 250 bp and 1 kb, presence of these bands suggests that isolates might have evolved from a common ancestor but due to geographical isolation fallowed by natural selection and genetic drift might have segregated in to subpopulations. Genetic similarity in the pathogenic population reflects the dispersal of single lineage in all locations in India.     

MucA-mediated coordination of type III secretion and alginate synthesis in Pseudomonas aeruginosa

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.     

Gelsolin and non-muscle myosin IIA interact to mediate calcium-regulated collagen phagocytosis

The formation of adhesion complexes is the rate-limiting step for collagen phagocytosis by fibroblasts, but the role of Ca(2+) and the potential interactions of actin-binding proteins in regulating collagen phagocytosis are not well defined. We found that the binding of collagen beads to fibroblasts was temporally and spatially associated with actin assembly at nascent phagosomes, which was absent in gelsolin null cells. Analysis of tryptic digests isolated from gelsolin immunoprecipitates indicated that non-muscle (NM) myosin IIA may bind to gelsolin. Immunostaining and immunoprecipitation showed that gelsolin and NM myosin IIA associated at collagen adhesion sites. Gelsolin and NM myosin IIA were both required for collagen binding and internalization. Collagen binding to cells initiated a prolonged increase of [Ca(2+)](i), which was absent in cells null for gelsolin or NM myosin IIA. Collagen bead-induced increases of [Ca(2+)](i) were associated with phosphorylation of the myosin light chain, which was dependent on gelsolin. NM myosin IIA filament assembly, which was dependent on myosin light chain phosphorylation and increased [Ca(2+)](i), also required gelsolin. Ionomycin-induced increases of [Ca(2+)](i) overcame the block of myosin filament assembly in gelsolin null cells. We conclude that gelsolin and NM myosin IIA interact at collagen adhesion sites to enable NM myosin IIA filament assembly and localized, Ca(2+)-dependent remodeling of actin at the nascent phagosome and that these steps are required for collagen phagocytosis.     

A role for gelsolin in stress fiber-dependent cell contraction.

Gelsolin is an abundant actin binding protein that mediates the rapid remodeling of cortical actin filaments through severing, capping, and nucleating activities. Most of the attention on the intracellular function of gelsolin has focused on the remodeling of the cortical actin meshwork but the localization of gelsolin to other regions of the cell suggests that it may have other important functions elsewhere. In cultured fibroblasts, gelsolin is heavily enriched in stress fibers, where its function in these contractile organelles is unknown. To study gelsolin function during stress fiber formation and cell contraction, we first assessed gelsolin levels in stress fiber preparations from lysophosphatidic acid (LPA)-treated human fibroblasts. LPA induced a large, time-dependent, calcium-independent increase of actin, gelsolin, alpha-actinin, and tropomyosin in stress fiber preparations. A microinjected gelsolin antibody that inhibits severing by gelsolin reduced stress fibers. Anti-sense-transfected gelsolin-depleted 3T3 cell lines treated with LPA after serum starvation required approximately 6 h to form stress fibers and focal adhesions, in contrast to control lines transfected with vector only, which formed stress fibers 15 min after addition of LPA. In cells microinjected with the gelsolin antibody that inhibits severing, Mg-ATP-induced cell contraction was greatly reduced in approximately 90% of injected cells compared to cells injected with an irrelevant antibody. Gelsolin-depleted cells were incapable of collagen gel contraction and showed no apparent Mg-ATP-induced cell contraction compared to cell lines transfected with vector only. The involvement of gelsolin in cell contraction and remodeling of collagen gels suggests a novel role for gelsolin in stress fiber-dependent cell function.     

Identification of specificity-defining amino acids of the wheat immune receptor Pm2 and powdery mildew effector AvrPm2

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) act as intracellular sensors for pathogen-derived effector proteins and trigger an immune response, frequently resulting in the hypersensitive cell death response (HR) of the infected host cell. The wheat (Triticum aestivum) NLR Pm2 confers resistance against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) if the isolate contains the specific RNase-like effector AvrPm2. We identified and isolated seven new Pm2 alleles (Pm2e–i) in the wheat D-genome ancestor Aegilops tauschii and two new natural AvrPm2 haplotypes from Bgt. Upon transient co-expression in Nicotiana benthamiana, we observed a variant-specific HR of the Pm2 variants Pm2a and Pm2i towards AvrPm2 or its homolog from the AvrPm2 effector family, BgtE-5843, respectively. Through the introduction of naturally occurring non-synonymous single nucleotide polymorphisms and structure-guided mutations, we identified single amino acids in both the wheat NLR Pm2 and the fungal effector proteins AvrPm2 and BgtE-5843 responsible for the variant-specific HR of the Pm2 variants. Exchanging these amino acids led to a modified HR of the Pm2–AvrPm2 interaction and allowed the identification of the effector head epitope, a 20-amino-acid long unit of AvrPm2 involved in the HR. Swapping of the AvrPm2 head epitope to the non-HR-triggering AvrPm2 family member BgtE-5846 led to gain of a HR by Pm2a. Our study presents a molecular approach to identify crucial effector surface structures involved in the HR and demonstrates that natural and induced diversity in an immune receptor and its corresponding effectors can provide the basis for understanding and modifying NLR–effector specificity.     

Stress modulates calcium mobilization in immune cells

Both acute and chronic restraint stress modulated mitogen-induced increases in cytoplasmic free-calcium concentrations ([Ca2+]i) in mouse spleen cells. Dual-color analysis of lymphocyte subpopulations demonstrated that acute (2 hour) restraint stress suppressed mitogen-stimulated increases in [Ca2+]i in CD4+ T cells, but enhanced [Ca2+]i in CD8+ T cells. Chronic restraint stress (2 hours daily for up to 21 days) resulted in a significant suppression of mitogen-stimulated increases in [Ca2+]i in CD4+ T cells at 3 and 7 days, but not at 21 days. CD8+ T cells were unaffected by chronic stress. Chronic stress (for 7 days) had a modest suppressive effect on mitogen-induced Ca2+ responses in B cells. Within T lymphocyte subpopulations, both acute and chronic stress predominantly affected CD4+ T cells, which may induce a functional reversal of the CD4/CD8 ratios in vivo. Such a reversal could result in suppression of a variety of immune responses such as lymphocyte proliferation and antigen-specific antibody production. These findings indicate that the inhibitory effects of stress on calcium mobilization in lymphocytes may be an early event mediating stress-induced immunosuppression.     

Glucose phosphorylation. Site-directed mutations which impair the catalytic function of hexokinase

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K.K., Fanciulli, M., and Pedersen, P.L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657----Ala mutation is the most profound, reducing the hexokinase activity to a level less than 1% of the wild type. The relative Vmax values for Ser-603----Ala, Glu-708----Ala, and Glu-742----Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603----Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558----Arg and Lys-558----Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in the apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.