Identification of Coffee Genes Expressed During Systemic Acquired Resistance and Incompatible Interaction with Hemileia vastatrix

Coffee genes associated with systemic acquired resistance (SAR) and incompatible reaction against coffee leaf rust inoculation were identified by suppression subtractive hybridization. Analysis of 384 clones of each of the subtracted cDNA libraries identified genes involved in oxidative burst/apoptosis/hypersensitive response, synthesis of antimicrobial proteins, synthesis and transport of antimicrobial metabolites, signal perception and transduction, metabolism of lipids, regulated protein degradation and cell maintenance and development. Induction of distinct sets of genes in the two resistance responses was observed. A wide range of genes involved in defence responses described in other plant species was also found in coffee plants. Semi-quantitative and quantitative RT-PCR analysis of seven selected genes showed differences in their expression profile within 72 h after treatment. Full-length cDNA sequences of two β-1,3-glucanases, one induced during SAR and the other in the incompatible reaction, were obtained by 5' and 3' RACE and the sequence data suggest different properties and cellular localization of the encoded proteins.     

Signal transduction in the wound response of tomato plants.

The wound response of tomato plants has been extensively studied, and provides a useful model to understand signal transduction events leading from injury to marker gene expression. The principal markers that have been used in these studies are genes encoding proteinase inhibitor (pin) proteins. Activation of pin genes occurs in the wounded leaf and in distant unwounded leaves of the plant. This paper reviews current understanding of signalling pathways in the wounded leaf, and in the systemically responding unwounded leaves. First, the nature of known elicitors and their potential roles in planta are discussed, in particular, oligogalacturonides, jasmonates and the peptide signal, systemin. Inhibitors of wound-induced proteinase inhibitor (pin) expression are also reviewed, with particular reference to phenolics, sulphydryl reagents and fusicoccin. In each section, results obtained from the bioassay are considered within the wider context of data from mutants and from transgenic plants with altered levels of putative signalling components. Following this introduction, current models for pin gene regulation are described and discussed, together with a summary for the involvement of phosphorylation-dephosphorylation in wound signalling. Finally, a new model for wound-induced pin gene expression is presented, arising from recent data from the author''s laboratory.     

Expression of biotin-binding proteins,avidin and streptavidin,in plant tissues using plant vacuolar targeting sequences

Tobacco plants have been developed which constitutively express high levels of the biotin-binding proteins, avidin and streptavidin. These plants were phenotypically normal and produced fertile pollen and seeds. The transgene was expressed and its product located in the vacuoles of most cell types in the plants. Targeting was achieved by use of N-terminal vacuolar targeting sequences derived from potato proteinase inhibitors which are known to target constitutively to vacuoles in potato tubers and, under wound-induction, in tomato leaves. Avidin was located in protein body-like structures within the vacuole and transgene protein levels remained relatively constant throughout the lifetime of the leaf. We describe two chimeric constructs with similar levels of expression. One comprised a potato proteinase inhibitor I signal peptide cDNA sequence attached to an avidin cDNA and the second a potato proteinase inhibitor II signal peptide genomic sequence (including an intron) attached to a core streptavidin synthetic sequence. We were unable to regenerate plants when transformation used constructs lacking the targeting sequences. The highest levels observed (up to 1.5% of total leaf protein) confirm the vacuole as the organelle of choice for stable storage of plant-toxic transgene products. The efficient targeting of these proteins did not result in any measured changes in plant biotinmetabolism.     

Wound- and systemin-inducible calmodulin gene expression in tomato leaves

Using a calmodulin (CaM) cDNA as a probe in northern analyses, transgenic tomato plants that overexpress the prosystemin gene were found to express increased levels of CaM mRNA and protein in leaves compared to wild-type plants. These transgenic plants have been reported previously to express several wound-inducible defense-related genes in the absence of wounding. Calmodulin mRNA and protein levels were found to increase in leaves of young wild-type tomato plants after wounding, or treatment with systemin, methyl jasmonate, or linolenic acid. CaM mRNA appeared within 0.5 h after wounding or supplying young tomato plants with systemin, and peaked at 1 h. The timing of CaM gene expression is similar to the expression of the wound- or systemin-induced lipoxygenase and prosystemin genes, signal pathway genes whose expression have been reported to begin at 0.5–1 h after wounding and 1–2 h earlier than the genes coding for defensive proteinase inhibitor genes. The similarities in timing between the synthesis of CaM mRNA and the mRNAs for signal pathway components suggests that CaM gene expression may be associated with the signaling cascade that activates defensive genes in response to wounding.     

Molecular cloning of a tomato leaf cDNA encoding an aspartic protease, a systemic wound response protein

A full-length cDNA encoding an aspartic protease (LeAspP) has been cloned from a tomato leaf cDNA library. Using LeAspP cDNA as a probe in gel blots, LeAspP mRNA was shown to be systemically induced in tomato leaves by wounding. Application of methyl jasmonate to leaves of intact tomato plants, or supplying systemin to young tomato plants through their cut stems, induces synthesis of LeAspP mRNA. LeAspP message is regulated in tomato similar to several systemic wound response proteins (swrps) that are part of the defense response in tomato plants directed against herbivore attacks.     

Systemic signaling in tomato plants for defense against herbivores. Isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene

An 18-amino acid peptide in tomato leaves called systemin is a primary signal released at wound sites in response to herbivory that systemically signals the activation of defense genes throughout the plants. We report here the isolation of three hydroxyproline-rich glycopeptides from tomato leaves, of 20, 18, and 15 amino acids in length, that signal the activation of defense genes, similar to the activity of the systemin peptide. The three new peptides cause an alkalinization of suspension-cultured cells and induce the synthesis of defensive proteinase inhibitor proteins when supplied at fmol levels to young tomato plants through their cut stems. This suggests that they are part of the wound signaling of tomato plants that activates defense against herbivores and pathogens. Isolation of cDNAs coding for the tomato peptides revealed that they are all derived from the same pre-proprotein precursor that is systemically wound-inducible. The peptides are considered members of the functionally characterized systemin family of defense signals from plants that are synthesized both in wounded leaves and in distal, unwounded leaves in response to herbivory or other mechanical wounding. The precursor deduced from the cDNA exhibits a leader sequence, indicating that it is synthesized through the secretory pathway, where it is hydroxylated and glycosylated. The amino acid sequence of the precursor exhibited weak identity to the precursor of two hydroxyproline-rich defense signals recently found in tobacco, suggesting that the two pre-protein precursors have evolved from a common ancestral protein. The identification of hydroxyproline-rich glycoprotein systemins in tomato indicates that the initiation of wound signaling is more complex than previously thought and appears to involve multiple peptide signals.     

Tobacco proteinase inhibitor I genes are locally,but not systemically induced by stress

A cDNA library of tobacco mosaic virus (TMV)-infected tobacco was screened with polymerase chain reaction products obtained using a degenerate primer corresponding to proteinase inhibitor I (PI-I) of tomato and potato. The resulting clones encoded two highly similar, putative tobacco PI-I proteins, indicating that both genes identified in tobacco are probably expressed. The tobacco PI-I's were approximately 50% identical to wound-inducible potato and tomato PI-I and 80% identical to an ethylene-regulated tomato PI-I. Northern blot analyses indicated that healthy tobacco leaf contains only minor amounts of PI-I mRNA, and that the inhibitor genes are induced by TMV infection, salicylate treatment, ethephon spraying, UV light irradiation and wounding. The results indicate that the tobacco PI-I genes are coordinately expressed with the genes for the basic pathogenesis-related proteins. Contrary to PI-I genes of tomato and potato, wound induction of the tobacco genes occurs only locally; the upper, unwounded leaves do not show any wound-induced PI-I gene expression.     

Chitinase accumulates systemically in wounded poplar trees

Young leaves of poplar ( Populus spp.) trees accumulate novel messenger RNAs shortly after the mature leaves have been mechanically wounded. These systemically wound‐induced ( win ) mRNAs are thought to encode proteins involved in plant defense. In the present paper, transgenic tobacco plants that ectopically expressed a win6 cDNA contained a novel chitinase activity that was not present in normal tobacco. This demonstrated Win6 was a chitinase. Win6 and a related protein Win8 accumulated in wounded poplars. Win6 and Win8 had low isoelectric points (ca 4) as predicted from their nucleotide sequence. The wound‐inducible increase in Win6 and Win8 was correlated with an increase in chitinase (EC 3.2.1.14) activity in poplar leaf extracts. We conclude that mechanical wounding induces chitinase in poplar trees, and speculate that the induced chitinase activity could act to increase the tolerance of poplars to opportunistic wound pathogens.