No abstract

The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well.

Genes encoding homologs of the gp91 phox subunit of the plasma membrane NADPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, the direct involvement of the gene products in superoxide (O 2 Ϫ ) production has yet to be shown. A novel activity gel assay based on protein fractionation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide gels was developed. In native polyacrylamide gel electrophoresis, one or two major O 2 Ϫ -producing formazan bands were detected in tomato (Lycopersicum esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsun, NN) plasma membranes, respectively. Denaturing fractionation of tomato and tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, followed by regeneration of the in-gel activity, revealed NADPH-dependent O 2 Ϫ -producing formazan bands of 106-, 103-, and 80-to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely inhibited by diphenylene iodonium or CuZn-O 2 Ϫ dismutase, indicating that the formazan precipitates were due to reduction by O 2 Ϫ radicals catalyzed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibody prepared from the C terminus of the tomato gp91 phox homolog. Membrane extracts as well as the in-gel NADPH oxidase activities were stimulated in the presence of Ca 2ϩ . In addition, the relative activity of the gp91 phox homolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp91 phox , the plant homolog can produce O 2 Ϫ in the absence of additional cytosolic components and is stimulated directly by Ca 2ϩ .

No abstract

SummaryAlternative splicing (AS) combines different transcript splice junctions that result in transcripts with shuffled exons, alternative 5¢ or 3¢ splicing sites, retained introns and different transcript termini. In this way, multiple mRNA species and proteins can be created from a single gene expanding the potential informational content of eukaryotic genomes. Search algorithms of AS forms in a variety of Arabidopsis databases showed they contained an unusually high fraction of retained introns (above 30%), compared with 10% that was reported for humans. The preponderance of retained introns (65%) were either part of open reading frames, present in the UTR region or present as the last intron in the transcript, indicating that their occurrence would not participate in non-sense-mediated decay. Interestingly, the functional distribution of the transcripts with retained introns is skewed towards stress and external/internal stimuli-related functions. A sampling of the alternative transcripts with retained introns were confirmed by RT-PCR and were shown to co-purify with polyribosomes, indicating their nuclear export. Thus, retained introns are a prominent feature of AS in Arabidopsis and as such may play a regulatory function.

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, pu-tatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epider-mal tissue. ROS, generated within cells, quickly moved apoplastically as H202 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the funga1 elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the externa1 addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase-or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluores-cein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.

The regulation of gene expression by cellular calcium is crucial for plant defense against biotic and abiotic stresses. However, the number of genes known to respond to specific transient calcium signals is limited, and as yet there is no definition of a calcium-responsive cis element in plants. Here, we generated specific cytosolic calcium transients in intact Arabidopsis thaliana seedlings and linked them to early transcriptome changes, followed by bioinformatic analysis of the responsive genes. A cytosolic calcium transient induced by calmodulin antagonists and blocked by lanthanides was characterized using aequorin-based luminometry and photon imaging. Analysis of transcriptome changes revealed 230 calcium-responsive genes, of which 162 were upregulated and 68 were downregulated. These include known early stress-responsive genes as well as genes of unknown function. Analysis of their upstream regions revealed, exclusively in the upregulated genes, a highly significant occurrence of a consensus sequence (P < 10−13) comprising two abscisic acid–specific cis elements: the abscisic acid–responsive element (ABRE; CACGTG[T/C/G]) and its coupling element ([C/A]ACGCG[T/C/A]). Finally, we show that a tetramer of the ABRE cis element is sufficient to confer transcriptional activation in response to cytosolic Ca2+ transients. Thus, at least for some specific Ca2+ transients and motif combinations, ABREs function as Ca2+-responsive cis elements.

Insidious fungal infections by postharvest pathogens remain quiescent during fruit growth until, at a particular phase during fruit ripening and senescence, the pathogens switch to the necrotrophic lifestyle and cause decay. During ripening, fruits undergo physiological processes, such as activation of ethylene biosynthesis, cuticular changes, and cell-wall loosening-changes that are accompanied by a decline of antifungal compounds, both those that are preformed and those that are inducible secondary metabolites. Pathogen infection of the unripe host fruit initiates defensive signal-transduction cascades, culminating in accumulation of antifungal proteins that limit fungal growth and development. In contrast, development of the same pathogens during fruit ripening and storage activates a substantially different signaling network, one that facilitates aggressive fungal colonization. This review focuses on responses induced by the quiescent pathogens of postharvest diseases in unripe host fruits. New genome-scale experimental approaches have begun to delineate the complex and multiple networks of host and pathogen responses activated to maintain or to facilitate the transition from the quiescent to the necrotrophic lifestyle.