An in planta induced gene of Phytophthora infestans codes for ubiquitin

An in planta induced gene of Phytophthora infestans (the causal organism of potato late blight) was selected from a genomic library by differential hybridization using labelled cDNA derived from poly(A)+ RNA of P. infestans grown in vitro and labelled cDNA made from potato-P. infestans interaction poly(A)+ RNA as probes. Sequence analysis showed that the gene codes for ubiquitin, a highly conserved protein which plays an important role in several cellular processes. The structure of the polyubiquitin gene (designated ubi3 R) is consistent with the structure of other known polyubiquitin genes. It consists of three repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extra asparagine residue at the carboxy-terminal end. Northern and Southern blot analyses revealed that the polyubiquitin gene is a member of a multigene family, all genes of which show induced expression in planta.     

Cloning,sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants

The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.     

Expression of the Phytophthora infestans ipiB and ipi0 genes in planta and in vitro

The ipiB and ipiO genes of the potato late blight fungus Phytophthora infestans (Mont.) de Bary were isolated from a genomic library in a screen for genes induced in planta. Expression of these genes was studied during pathogenesis on various host tissues and different host plants, some of which show specific resistance against P. infestans infection. During pathogenesis on leaves and tubers of the fully susceptible potato cultivar (cv.) Ajax and on leaves of the fully susceptible tomato cv. Moneymaker, the P. infestans ipiB and ipiO genes show a transient expression pattern with highest mRNA levels in the early stages of infection. During the interaction with leaves of the partially resistant potato cv. Pimpernel, the expression is also transient but accumulation and disappearance of the mRNAs is delayed. Also in P. infestans inoculated onto a race-specific resistant potato cultivar and onto the nonhost Solanum nigrum, ipiB and ipiO mRNA is detectable during the initial stages of infection. Apparently, the expression of the ipiB and the ipiO genes is activated in compatible, incompatible and nonhost interactions. In encysted zoospores, ipiB and ipiO mRNA accumulation was not detectable, but during cyst germination and appressorium formation on an artificial surface the genes are highly expressed. Expression studies in mycelium grown in vitro revealed that during nutrient starvation the expression of the ipiB and ipiO genes is induced. For ipiO gene expression, carbon deprivation appeared to be sufficient. The ipiO gene promoters contain a sequence motif that functions as a glucose repression element in yeast and this motif might be involved in the regulation of ipiO gene expression.     

Comparative structural and functional analysis of genes encoding pectin methylesterases in Phytophthora spp.

We have scanned the Phytophthora infestans, P. ramorum, and P. sojae genomes for the presence of putative pectin methylesterase genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. infestans models, and investigated the gene expression levels throughout the course of P. infestans infection on potato plants, using in planta and detached leaf assays. We found that genes located on contiguous chromosomal regions contain similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Results of our investigations also suggest that, during the pathogenicity process, the expression levels of some of the analyzed genes vary considerably when compared to basal expression observed in in vitro cultures of non-sporulating mycelium. These results were observed both in planta and in detached leaf assays.     

Analysis of late-blight disease resistance and freezing tolerance in transgenic potato plants expressing sense and antisense genes for an osmotin-like protein

The expression patterns of plant defense genes encoding osmotin and osmotin-like proteins imply a dual function in osmotic stress and plant pathogen defense. We have produced transgenic potato (Solanum commersonii Dun.) plants constitutively expressing sense or antisense RNAs from chimeric gene constructs consisting of the cauliflower mosaic virus 35S promoter and a cDNA (pA13) for an osmotin-like protein. Transgenic potato plants expressing high levels of the pA13 osmotin-like protein showed an increased tolerance to the late-blight fungus Phytophthora infestans at various phases of infection, with a greater resistance at an early phase of fungal infection. There was a decrease in the accumulation of osmotin-like mRNAs and proteins when antisense transformants were challenged by fungal infection, although the antisense transformants did not exhibit any alterations in disease susceptibility. Expression of pA13 sense and antisense RNAs had no effect on the development of freezing tolerance in transgenic plants when assayed under a variety of conditions including treatments with abscisic acid or low temperature. These results provide evidence of antifungal activity for a potato osmotin-like protein against the fungus P. infestans, but do not indicate that pA13 osmotin-like protein is a major determinant of freezing tolerance.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.     

The infection cushion of Botrytis cinerea: a fungal ‘weapon’ of plant-biomass destruction

The necrotrophic plant-pathogen fungus Botrytis cinerea produces multicellular appressoria dedicated to plant penetration, named infection cushions (IC). A microarray analysis was performed to identify genes upregulated in mature IC. The expression data were validated by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and biochemical assays. 1231 upregulated genes and 79 up-accumulated proteins were identified. The data support the secretion of effectors by IC: phytotoxins, ROS, proteases, cutinases, plant cell wall–degrading enzymes and plant cell death–inducing proteins. Parallel upregulation of sugar transport and sugar catabolism–encoding genes would indicate a role of IC in nutrition. The data also reveal a substantial remodelling of the IC cell wall and suggest a role for melanin and chitosan in IC function. Lastly, mutagenesis of two upregulated genes in IC identified secreted fasciclin-like proteins as actors in the pathogenesis of B. cinerea. These results support the role of IC in plant penetration and also introduce other unexpected functions for this fungal organ, in colonization, necrotrophy and nutrition of the pathogen.     

Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.