Identification of drought-response genes and a study of their expression during sucrose accumulation and water deficit in sugarcane culms.

BACKGROUND: The ability of sugarcane to accumulate high concentrations of sucrose in its culm requires adaptation to maintain cellular function under the high solute load. We have investigated the expression of 51 genes implicated in abiotic stress to determine their expression in the context of sucrose accumulation by studying mature and immature culm internodes of a high sucrose accumulating sugarcane cultivar. Using a sub-set of eight genes, expression was examined in mature internode tissues of sugarcane cultivars as well as ancestral and more widely related species with a range of sucrose contents. Expression of these genes was also analysed in internode tissue from a high sucrose cultivar undergoing water deficit stress to compare effects of sucrose accumulation and water deficit. RESULTS: A sub-set of stress-related genes that are potentially associated with sucrose accumulation in sugarcane culms was identified through correlation analysis, and these included genes encoding enzymes involved in amino acid metabolism, a sugar transporter and a transcription factor. Subsequent analysis of the expression of these stress-response genes in sugarcane plants that were under water deficit stress revealed a different transcriptional profile to that which correlated with sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit but this did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis indicated that proline was negatively correlated with sucrose concentration, and whilst total amino acid concentrations increased about seven-fold under water deficit, the relatively low concentration of proline suggested that it had no osmoprotectant role in sugarcane culms. CONCLUSIONS: The results show that while there was a change in stress-related gene expression associated with sucrose accumulation, different mechanisms are responding to the stress induced by water deficit, because different genes had altered expression under water deficit.

Identification and occurrence of the LTR-Copia-like retrotransposon, PSCR and other Copia-like elements in the genome of Phytophthora sojae.

Sequence analysis of the genomic region of Phytophthora sojae close to the Avr4/6 locus specifying virulence on soybean identified a Ty1/Copia-like retrotransposon that we have named Phytophthora sojae Copia-like retrotransposon (PSCR). Twelve near-complete homologs of PSCR were found in the published P. sojae genome sequence, none of which encoded a full-length polyprotein characteristic of Copia-like retrotransposons, or appears to exhibit transcriptional activity or show evidence of recent movement, suggesting they are non-functional and unlikely to have caused pathogenic variability. However, reconstructed consensus PSCR sequence encoding a full-length polyprotein resembles a functional, ancestral retroelement within P. sojae. Homologs were also found in sequence databases of other Phytophthora species. Database searches found other families of Copia-like elements in genomes of P. sojae, P. ramorum and P. infestans that were different from members of the PSCR family and from Copia-like elements reported in other organisms. It is possible that the various families of Copia-like retroelements identified in this study represent introgressions into the genome of ancient ancestor(s) of current Phytophthora species, where they have evolved and diverged considerably during the speciation. Some Copia-like families are transcriptionally active with the potential to transpose and contribute to pathogenic variation in current populations of P. sojae.

The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat.

Fusarium species infect cereal crops worldwide and cause the important diseases Fusarium head blight and crown rot in wheat. Fusarium pathogens reduce yield and some species also produce trichothecene mycotoxins, such as deoxynivalenol (DON), during infection. These toxins play roles in pathogenesis on wheat and have serious health effects if present in grain consumed by humans or animals. In the present study, the response of wheat tissue to DON has been investigated. Infusion of wheat leaves with DON induced hydrogen peroxide production within 6 h followed by cell death within 24 h that was accompanied by DNA laddering, a hallmark of programmed cell death. In addition, real-time PCR analysis revealed that DON treatment rapidly induced transcription of a number of defence genes in a concentration-dependent manner. Co-treatment with DON and the antioxidant ascorbic acid reduced these responses, suggesting their induction may be at least partially mediated by reactive oxygen species (ROS), commonly known to be signalling molecules in plants. Wheat defence genes were more highly expressed in wheat stems inoculated with a DON-producing fungal strain than those inoculated with a DON-non-producing mutant, but only at a late stage of infection. Taken together, the results are consistent with a model in which DON production during infection of wheat induces ROS, which on the one hand may stimulate programmed host cell death assisting necrotrophic fungal growth, whereas, on the other hand, the ROS may contribute to the induction of antimicrobial host defences.

A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole.

Sugarcane is an ideal candidate as a biofactory for the production of alternate higher value products. One way of achieving this is to direct useful proteins into the vacuoles within the sugarcane storage parenchyma tissue. By bioinformatic analysis of gene sequences from putative sugarcane vacuolar proteins a motif has been identified that displays high conservation across plant legumain homologues that are known to function within vacuolar compartments. This five amino acid motif, represented by the sequence IRLPS in sugarcane is shown to direct an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP appeared to be dependent on cell type, suggesting that the vacuolar environment can be hostile to introduced proteins. This targeting motif will be a valuable tool for engineering plants such as sugarcane for production of novel products.

Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum. Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

The SEN1 gene of Arabidopsis is regulated by signals that link plant defence responses and senescence.

Plant defence and senescence share many similarities as evidenced by extensive co-regulation of many genes during these responses. To better understand the nature of signals that are common to plant defence and senescence, we studied the regulation of SEN1 encoding a senescence-associated protein during plant defence responses in Arabidopsis. Pathogen inoculations and treatments with defence-related chemical signals, salicylic acid and methyl jasmonate induced changes in SEN1 transcript levels. Analysis of transgenic plants expressing the SEN1 promoter fused to uidA reporter gene confirmed the responsiveness of the SEN1 promoter to defence- and senescence-associated signals. Expression analysis of SEN1 in a number of defence signalling mutants indicated that activation of this gene by pathogen occurs predominantly via the salicylic and jasmonic acid signalling pathways, involving the functions of EDS5, NPR1 and JAR1. In addition, in the absence of pathogen challenge, the cpr5/hys1 mutant showed elevated SEN1 expression and displayed an accelerated senescence response following inoculation with the necrotrophic fungal pathogen Fusarium oxysporum. Although the analysis of the sen1-1 knock-out mutant did not reveal any obvious role for this gene in defence or senescence-associated events, our results presented here show that SEN1 is regulated by signals that link plant defence and senescence responses and thus represents a useful marker gene to study the overlap between these two important physiological events.

CgDN24: a gene involved in hyphal development in the fungal phytopathogen Colletotrichum gloeosporioides.

A cDNA corresponding to a transcript induced in culture by N starvation, was identified in Colletotrichum gloeosporioides by a differential hybridisation strategy. The cDNA comprised 905 bp and predicted a 215 aa protein; the gene encoding the cDNA was termed CgDN24. No function for CgDN24 could be predicted by database homology searches using the cDNA sequence and no homologues were found in the sequenced fungal genomes. Transcripts of CgDN24 were detected in infected leaves of Stylosanthes guianensis at stages of infection that corresponded with symptom development. The CgDN24 gene was disrupted by homologous recombination and this led to reduced radial growth rates and the production of hyphae with a hyperbranching phenotype. Normal sporulation was observed, and following conidial inoculation of S. guianensis, normal disease development was obtained. These results demonstrate that CgDN24 is necessary for normal hyphal development in axenic culture but dispensable for phytopathogenicity.

Repressor- and activator-type ethylene response factors functioning in jasmonate signaling and disease resistance identified via a genome-wide screen of Arabidopsis transcription factor gene expression.

To identify transcription factors (TFs) involved in jasmonate (JA) signaling and plant defense, we screened 1,534 Arabidopsis (Arabidopsis thaliana) TFs by real-time quantitative reverse transcription-PCR for their altered transcript at 6 h following either methyl JA treatment or inoculation with the incompatible pathogen Alternaria brassicicola. We identified 134 TFs that showed a significant change in expression, including many APETALA2/ethylene response factor (AP2/ERF), MYB, WRKY, and NAC TF genes with unknown functions. Twenty TF genes were induced by both the pathogen and methyl JA and these included 10 members of the AP2/ERF TF family, primarily from the B1a and B3 subclusters. Functional analysis of the B1a TF AtERF4 revealed that AtERF4 acts as a novel negative regulator of JA-responsive defense gene expression and resistance to the necrotrophic fungal pathogen Fusarium oxysporum and antagonizes JA inhibition of root elongation. In contrast, functional analysis of the B3 TF AtERF2 showed that AtERF2 is a positive regulator of JA-responsive defense genes and resistance to F. oxysporum and enhances JA inhibition of root elongation. Our results suggest that plants coordinately express multiple repressor- and activator-type AP2/ERFs during pathogen challenge to modulate defense gene expression and disease resistance.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs.

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H(+)-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

The mode of action of the plant antimicrobial peptide MiAMP1 differs from that of its structural homologue, the yeast killer toxin WmKT.

The plant antimicrobial peptide MiAMP1 from Macadamia integrifolia and the yeast killer toxin peptide WmKT from Williopsis mrakii are structural homologues. Comparative studies of yeast mutants were performed to test their sensitivity to these two antimicrobial peptides. No differences in susceptibility to MiAMP1 were detected between wild-type and several WmKT-resistant mutant yeast strains. A yeast mutant MT1, resistant to MiAMP1 but unaffected in its susceptibility to plant defensins and hydrogen peroxide, also did not show enhanced tolerance towards WmKT. It is therefore probable that the Greek key beta-barrel structure shared by MiAMP1 and WmKT provides a robust structural framework ensuring stability for the two proteins but that the specific action of the peptides depends on other motifs.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis.

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Quantitative analysis of the expression of ACAT genes in human tissues by real-time PCR.

ACAT (also called sterol o-acyltransferase) catalyzes the esterification of cholesterol by reaction with long-chain acyl-CoA derivatives and plays a pivotal role in the regulation of cholesterol homeostasis. Although two human ACAT genes termed ACAT-1 and ACAT-2 have been reported, prior research on differential tissue expression is qualitative and incomplete. We have developed a quantitative multiplex assay for each ACAT isoform after RT treatment of total RNA using TaqMan real-time quantitative PCR normalized to beta-actin in the same reaction tube. This enabled us to calculate the relative abundance of transcripts in several human tissues as an ACAT-2/ACAT-1 ratio. In liver (n = 17), ACAT-1 transcripts were on average 9-fold (range, 1.7- to 167-fold) more abundant than ACAT-2, whereas in duodenal samples (n = 10), ACAT-2 transcripts were on average 3-fold (range, 0.39- to 12.2-fold) more abundant than ACAT-1. ACAT-2 was detected for the first time in peripheral blood mononuclear cells. Interesting differences in ACAT-2 mRNA expression were evident in subgroup analysis of samples from different sources. These results demonstrate quantitatively that ACAT-1 transcripts predominate in human liver and ACAT-2 transcripts predominate in human duodenum and support the notion that ACAT-2 has an important regulatory role in liver and intestine.

Phytophthora sojae avirulence genes Avr4 and Avr6 are located in a 24kb, recombination-rich region of genomic DNA.

A cross between two different races (race 7xrace 25) of the soybean root and stem rot pathogen Phytophthora sojae was analyzed to characterize the genomic region flanking two cosegregating avirulence genes, Avr4 and Avr6. Both genes cosegregated in the ratio of 82:17 (avirulent:virulent) in an F(2) population, suggestive of a single locus controlling both phenotypes. A chromosome walk was commenced from RAPD marker OPE7.1C, 2.0cM distant from the Avr4/6 locus. Three overlapping cosmids were isolated which included genetic markers that flank the Avr4/6 locus. The chromosome walk spanned a physical distance of 67kb which represented a genetic map distance of 22.3cM, an average recombination frequency of 3.0kb/cM and 11.7-fold greater than the predicted average recombination frequency of 35.3kb/cM for the entire P. sojae genome. Six genes (cDNA clones) expressed from the Avr4/6 genomic region encompassed by the cosmid contig were identified. Single nucleotide polymorphisms and restriction fragment length polymorphisms showed these six genes were closely linked to the Avr4/6 locus. Physical mapping of the cDNA clones within the cosmid contig made it possible to deduce the precise linkage order of the cDNAs. None of the six cDNA clones appear to be candidates for Avr4/6. We conclude that two of these cDNA clones flank a physical region of approximately 24kb and 4.3cM that appears to include the Avr4/6 locus.