Molecular Plant-Microbe Interactions

We show that the cyanobacterial symbionts of a tripartite cyanolichen can produce hepatotoxic microcystins in situ. Microcystins were detected with high-performance liquid chromatography mass spectrometry both from cephalodia of the tripartite cyanolichen Peltigera leucophlebia and from a symbiotic Nostoc strain isolated from the same lichen specimen. Genetic identities of symbiotic Nostoc strains were studied by amplifying and sequencing the 16S rRNA gene. Also, the presence of the microcystin synthetase gene mcyE was confirmed by sequencing. Three highly toxic microcystins were detected from the lichen specimen. Several different Nostoc 16S rRNA haplotypes were present in the lichen sample but only one was found in the toxin-producing cultures. In culture, the toxin-producing Nostoc strain produced a total of 19 different microcystin variants. In phylogenetic analysis, this cyanobacterium and related strains from the lichen thallus grouped together with a previously known microcystin-producing Nostoc strain and other strains previously isolated from the symbiotic thalloid bryophyte Blasia pusilla. Our finding is the first direct evidence of in situ production of microcystins in lichens or plant–cyanobacterial symbioses. Microcystins may explain why cyanolichens and symbiotic bryophytes are not among the preferred food sources of most animal grazers.

The cell-to-cell and long-distance movement of the bipartite geminivirus, bean dwarf mosaic (BDMV), in Phaseolus vulgaris plants was examined with the noninvasive reporter, the green fluorescent protein (GFP). A modified GFP gene (mGFP4) was inserted into the BDMV DNA-A component in place of the coat protein gene (BDMVA-mGFP4), and particle bombardment was used to introduce viral DNA into bean seedlings (radicle and hypocotyl tissues). Fluorescence analysis of GFP expressed from BDMVA-mGFP4 established that particle bombardment introduced viral DNA only into epidermal cells, and the requirement for the DNA-B-encoded proteins (BV1 and BC1) in the cell-to-cell movement of BDMVA-mGFP4. This GFP reporter system was used to follow the viral infection process from the seedling stage throughout the entire plant life cycle. In inoculated hypocotyls, BDMV moved from cell to cell through the cortex and showed a striking phloem tropism. Upon entry into phloem tissues, BDMV moved rapidly toward the root via the long-distance transport system, and toward the shoot apex by a combination of cell-to-cell and long-distance movement. Analysis of GFP distribution in systemically infected tissues revealed that BDMV was restricted to phloem cells in both roots and stems. In systemically infected primary and trifoliolate leaves, BDMV infected phloem cells associated with all vein orders (first through fifth), and the capacity of BDMV to exit from phloem tissue into nonphloem cells was correlated with the stage of plant development. Finally, fluorescence analysis of GFP in reproductive tissues established that BDMV infected flower, pod, and seed-coat tissues, but was excluded from the embryo.

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

In Sinorhizobium meliloti the NolR repressor displays differential negative regulation of nodulation genes and is required for optimal nodulation. Here, we demonstrate that the NolR function is not unique to S. meliloti but is also present in other species of the Rhizobiaceae family. DNA hybridization indicates the presence of nolR homologous sequences in species belonging to the Rhizobium and Sinorhizobium genera while no hybridization signal was detected in species from the Mesorhizobium, Bradyrhizo-bium, Azorhizobium, and Agrobacterium genera. We isolated the nolR gene from the Rhizobium leguminosarum bv. viciae strain TOM and showed that the TOM nolR gene acts similarly to S. meliloti nolR by repressing the expression of both the nodABCIJ and the nodD genes, resulting in decreased Nod factor production. The presence of a functional nolR gene in R. leguminosarum is correlated with an increased rate and extent of nodulation of pea. The conserved primary structure, the location of the DNA-binding domain, and the similar size of NolR proteins, compared with a family of small bacterial regulatory proteins including HlyU, SmtB, and the ArsR-type regulators, revealed that NolR belongs to this family.

Despite the fact that a large number of miRNA sequences have been determined in diverse plant species, reports demonstrating the functional roles of miRNAs in the plant response to pathogens are severely limited. Here, Arabidopsis thaliana miRNA844 (miR844) was investigated for its functional role in the defense response to diverse pathogens. Transgenic Arabidopsis plants overexpressing miR844 (35S::miR844) displayed much more severe disease symptoms than the wild-type plants when challenged with the bacterium Pseudomonas syringae pv. tomato DC3000 or the fungus Botrytis cinerea. By contrast, a loss-of-function mir844 mutant showed an enhanced resistance against the pathogens. Although no cleavage was observed at the predicted cleavage site of the putative target mRNA, cytidinephosphate diacylglycerol synthase3 (CDS3), cleavage was observed at 6, 12, 21, or 52 bases upstream of the predicted cleavage site of CDS3 mRNA, and the level of CDS3 mRNA was downregulated by the overexpression of miR844, implying that miR844 influences CDS3 transcript level. To further confirm that the miR844-mediated defense response was due to the decrease in CDS3 mRNA level, the disease response of a CDS3 loss-of-function mutant was analyzed upon pathogen challenge. Increased susceptibility of both cds3 mutant and 35S::miR844 plants to pathogens confirmed that miR844 affected the defense response by downregulating CDS3 mRNA. The expression of miR844 was decreased, and the CDS3 transcript level increased upon pathogen challenge. Taken together, these results provide evidence that downregulation of miR844 and a concomitant increase in CDS3 expression is a defensive response of Arabidopsis to bacteria and fungi.