Inducible expression of p50 from TMV for increased resistance to bacterial crown gall disease in tobacco.

The dominant tobacco mosaic virus (TMV) resistance gene N induces a hypersensitive response upon TMV infection and protects tobacco against systemic spread of the virus. It has been proposed to change disease resistance specificity by reprogramming the expression of resistance genes or their corresponding avirulence genes. To reprogramme the resistance response of N towards bacterial pathogens, the helicase domain (p50) of the TMV replicase, the avirulence gene of N, was linked to synthetic promoters 4D and 2S2D harbouring elicitor-responsive cis-elements. These promoter::p50 constructs induce local necrotic lesions on NN tobacco plants in an Agrobacterium tumefaciens infiltration assay. A tobacco genotype void of N (nn) was transformed with the promoter::p50 constructs and subsequently crossed to NN plants. Nn F1 offspring selected for the T-DNA develop normally under sterile conditions. After transfer to soil, some of the F1 plants expressing the 2S2D::p50 constructs develop spontaneous necrosis. Transgenic Nn F1 plants with 4D::p50 and 2S2D::p50 expressing constructs upregulate p50 transcription and induce local necrotic lesions in an A. tumefaciens infiltration assay. When leaves and stems of Nn F1 offspring harbouring promoter::p50 constructs are infected with oncogenic A. tumefaciens C58, transgenic lines harbouring the 2S2D::p50 construct induce necrosis and completely lack tumor development. These results demonstrate a successful reprogramming of the viral N gene response against bacterial crown gall disease and highlight the importance of achieving tight regulation of avirulence gene expression and the control of necrosis in the presence of the corresponding resistance gene.

CbCTB2, an O-methyltransferase is essential for biosynthesis of the phytotoxin cercosporin and infection of sugar beet by Cercospora beticola.

BACKGROUND: Cercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beets (Beta vulgaris) worldwide. Cercosporin, a light-inducible toxin, is essential for necrosis of the leaf tissue and development of the typical leaf spots on sugar beet leaves. RESULTS: In this study we show that the O-methyltransferase gene CTB2 is essential for cercosporin production and pathogenicity in two C. beticola isolates. We established a transformation system for C. beticola protoplasts, disrupted CTB2, and transformed the Δctb2 strains as well as a wild type strain with the DsRed reporter gene. The Δctb2 strains had lost their pigmentation and toxin measurements demonstrated that the Δctb2 strains were defective in cercosporin production. Infection of sugar beets with the wild type and Δctb2 DsRed strains showed that the deletion strain was severely impaired in plant infection. Histological analysis revealed that the CTB2-deficient isolate cannot enter the leaf tissue through stomata like the wild type. CONCLUSIONS: Taken together, these observations indicate that cercosporin has a dual function in sugar beet infection: in addition to the well-known role in tissue necrosis, the toxin is required for the early phase of sugar beet infection.

Integration of bioinformatics and synthetic promoters leads to the discovery of novel elicitor-responsive cis-regulatory sequences in Arabidopsis.

A combination of bioinformatic tools, high-throughput gene expression profiles, and the use of synthetic promoters is a powerful approach to discover and evaluate novel cis-sequences in response to specific stimuli. With Arabidopsis (Arabidopsis thaliana) microarray data annotated to the PathoPlant database, 732 different queries with a focus on fungal and oomycete pathogens were performed, leading to 510 up-regulated gene groups. Using the binding site estimation suite of tools, BEST, 407 conserved sequence motifs were identified in promoter regions of these coregulated gene sets. Motif similarities were determined with STAMP, classifying the 407 sequence motifs into 37 families. A comparative analysis of these 37 families with the AthaMap, PLACE, and AGRIS databases revealed similarities to known cis-elements but also led to the discovery of cis-sequences not yet implicated in pathogen response. Using a parsley (Petroselinum crispum) protoplast system and a modified reporter gene vector with an internal transformation control, 25 elicitor-responsive cis-sequences from 10 different motif families were identified. Many of the elicitor-responsive cis-sequences also drive reporter gene expression in an Agrobacterium tumefaciens infection assay in Nicotiana benthamiana. This work significantly increases the number of known elicitor-responsive cis-sequences and demonstrates the successful integration of a diverse set of bioinformatic resources combined with synthetic promoter analysis for data mining and functional screening in plant-pathogen interaction.

Transcript profiles in sugar beet genotypes uncover timing and strength of defense reactions to Cercospora beticola infection.

Cercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beet (Beta vulgaris) worldwide. Despite the great agronomical importance of this disease, little is known about its underlying molecular processes. Technical resources are scarce for analyzing this important crop species. We developed a sugar beet microarray with 44,000 oligonucleotides that represent 17,277 cDNAs. During the four stages of C. beticola-B. vulgaris interactions, we profiled the transcriptional responses of three genotypes: susceptible, polygenic partial resistance, and monogenic resistant. Similar genes were induced in all three genotypes during infection but with striking differences in timing. The monogenic resistant genotype displayed strong defense responses at 1 day postinoculation (dpi). The other genotypes displayed defense responses in a later phase (15 dpi) of the infection cycle. The partially resistant genotype displayed a strong defense response in the late phase of the infection cycle. Furthermore, the partially resistant genotype expressed pathogen-related transcripts that the susceptible genotype lacked. These results indicate that resistance was achieved by the ability to mount an early defense response, and partial resistance was determined by additional defense and signaling transcripts that allowed effective defense in the late phase of the infection cycle.

Accumulation of the hormone abscisic acid (ABA) at the infection site of the fungus Cercospora beticola supports the role of ABA as a repressor of plant defence in sugar beet.

Inducible plant defence responses in sugar beet (Beta vulgaris L.) leaves are repressed during the early phase of infection by the fungus Cercospora beticola. In this report, we show that the concentration of the plant hormone abscisic acid (ABA) increases in sugar beet leaves during C. beticola infection. After an initial burst of ABA induced by inoculation of the fungus, elevated ABA concentrations were detected during the fungal penetration and colonization phases 3-9 days after inoculation. Fifteen days after inoculation, with visible onset of the necrotic phase of infection, the strongly elevated ABA concentrations in infected leaves were at levels similar to drought-stressed plants. A synthetic promoter composed of four copies of the ABA-responsive element (ABRE) A2 and the coupling element CE3 of the ABA-inducible barley gene HVA1 was strongly induced by ABA and C. beticola infection in transgenic sugar beet leaves. Analysis of the spatial pattern of promoter activity revealed that the ABA-inducible promoter was locally activated at the fungal infection sites. Furthermore, expression of the basic leucine zipper transcription factor AREB1 was induced by drought stress and fungal infection in the sugar beet. Application of ABA reduced the promoter activity of the phenylalanine ammonia lyase (BvPAL) gene, and this effect was observed with the -34 to +248 BvPAL promoter region. This region is equivalent to the core promoter, which is necessary for the suppression of BvPAL expression by C. beticola, as recently shown. These data indicate that ABA accumulation and activation of the ABA-dependent signalling cascade are the primary cause of suppression of BvPAL expression during infection of sugar beet leaves.

Post-harvest regulated gene expression and splicing efficiency in storage roots of sugar beet (Beta vulgaris L.).

Sixteen post-harvest upregulated genes from sugar beet comprising five novel sequences were isolated by subtractive cloning. Transcription profiles covering a period of up to 49 days after harvest under controlled storage conditions and in field clamps are reported. Post-harvest induced genes are involved in wound response, pathogen defense, dehydration stress, and detoxification of reactive oxygen species. An early induction of a cationic peroxidase indicates a response to post-harvest damage. Wound response reactions may also involve genes required for cell division such as a regulator of chromatin condensation and a precursor of the growth stimulating peptide phytohormone phytosulfokine-alpha. Surprisingly, also three putative non-protein coding genes were isolated. Two of these genes show intron specific and storage temperature dependent splicing of a precursor mRNA. The temperature dependent splicing of an intron containing sugar beet mRNA is also maintained in transgenic Arabidopsis thaliana. The storage induced genes are integrated into a model that proposes the response to several post-harvest stress conditions. Temperature regulated splicing may be a mechanism to sense seasonal temperature changes.

Phytotoxicity and innate immune responses induced by Nep1-like proteins.

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)-like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

Taproot promoters cause tissue specific gene expression within the storage root of sugar beet.

The storage root (taproot) of sugar beet (Beta vulgaris L.) originates from hypocotyl and primary root and contains many different tissues such as central xylem, primary and secondary cambium, secondary xylem and phloem, and parenchyma. It was the aim of this work to characterize the promoters of three taproot-expressed genes with respect to their tissue specificity. To investigate this, promoters for the genes Tlp, His1-r, and Mll were cloned from sugar beet, linked to reporter genes and transformed into sugar beet and tobacco. Reporter gene expression analysis in transgenic sugar beet plants revealed that all three promoters are active in the storage root. Expression in storage root tissues is either restricted to the vascular zone (Tlp, His1-r) or is observed in the whole organ (Mll). The Mll gene is highly organ specific throughout different developmental stages of the sugar beet. In tobacco, the Tlp and Mll promoters drive reporter gene expression preferentially in hypocotyl and roots. The properties of the Mll promoter may be advantageous for the modification of sucrose metabolism in storage roots.

Suppression of phenylalanine ammonia lyase expression in sugar beet by the fungal pathogen Cercospora beticola is mediated at the core promoter of the gene.

The suppression of plant defence reactions plays a crucial role in causing plant diseases. In this report, we show that inducible plant defences are repressed during the development of Cercospora leaf spot disease. In the early phase of infection of sugar beet (Beta vulgaris L.) leaves with the phytopathogenic fungus Cercospora beticola , a reduction in the expression of the phenylalanine ammonia lyase (BvPAL) and cinnamic acid 4-hydroxylase (BvC4H) genes was observed. BvPAL reduction was found at the transcript and enzyme activity levels. In order to analyse the signal transduction process responsible for suppression, the BvPAL promoter was isolated. An abbreviated 5'- and 3'- deletion series of the promoter was effected using transient biolistic assays, which showed that the activity of a truncated promoter from positions -34 to +246, relative to the transcriptional starting site, retains approximately 30 of the activity of the full-length promoter. The region within the BvPAL promoter required for the reduction in transcription was identified as being positions -34 to +45, with respect to the start of the transcription. This region is equivalent to the core promoter, characterised by the TATA-box, an initiator (Inr) and an unknown downstream element in the region between +7 and +45. These data indicate that (1) plant defence responses are repressed during the development of Cercospora leaf spot disease and (2) the PAL core promoter is involved in the detection of the repression signal.

A sugar beet chlorophyll a/b binding protein promoter void of G-box like elements confers strong and leaf specific reporter gene expression in transgenic sugar beet.

BACKGROUND: Modification of leaf traits in sugar beet requires a strong leaf specific promoter. With such a promoter, expression in taproots can be avoided which may otherwise take away available energy resources for sugar accumulation. RESULTS: Suppression Subtractive Hybridization (SSH) was utilized to generate an enriched and equalized cDNA library for leaf expressed genes from sugar beet. Fourteen cDNA fragments corresponding to thirteen different genes were isolated. Northern blot analysis indicates the desired tissue specificity of these genes. The promoters for two chlorophyll a/b binding protein genes (Bvcab11 and Bvcab12) were isolated, linked to reporter genes, and transformed into sugar beet using promoter reporter gene fusions. Transient and transgenic analysis indicate that both promoters direct leaf specific gene expression. A bioinformatic analysis revealed that the Bvcab11 promoter is void of G-box like regulatory elements with a palindromic ACGT core sequence. The data indicate that the presence of a G-box element is not a prerequisite for leaf specific and light induced gene expression in sugar beet. CONCLUSIONS: This work shows that SSH can be successfully employed for the identification and subsequent isolation of tissue specific sugar beet promoters. These promoters are shown to drive strong leaf specific gene expression in transgenic sugar beet. The application of these promoters for expressing resistance improving genes against foliar diseases is discussed.

Construction of a 'unigene' cDNA clone set by oligonucleotide fingerprinting allows access to 25 000 potential sugar beet genes.

Access to the complete gene inventory of an organism is crucial to understanding physiological processes like development, differentiation, pathogenesis, or adaptation to the environment. Transcripts from many active genes are present at low copy numbers. Therefore, procedures that rely on random EST sequencing or on normalisation and subtraction methods have to produce massively redundant data to get access to low-abundance genes. Here, we present an improved oligonucleotide fingerprinting (ofp) approach to the genome of sugar beet (Beta vulgaris), a plant for which practically no molecular information has been available. To identify distinct genes and to provide a representative 'unigene' cDNA set for sugar beet, 159 936 cDNA clones were processed utilizing large-scale, high-throughput data generation and analysis methods. Data analysis yielded 30 444 ofp clusters reflecting the number of different genes in the original cDNA sample. A sample of 10 961 cDNA clones, each representing a different cluster, were selected for sequencing. Standard sequence analysis confirmed that 89% of these EST sequences did represent different genes. These results indicate that the full set of 30 444 ofp clusters represent up to 25 000 genes. We conclude that the ofp analysis pipeline is an accurate and effective way to construct large representative 'unigene' sets for any plant of interest with no requirement for prior molecular sequence data.

Isolation and molecular analysis of six taproot expressed genes from sugar beet.

The taproot from sugar beet (Beta vulgaris L.) undergoes a specific developmental process to function as a food storage organ. Suppression Subtractive Hybridization (SSH) was utilized for the isolation of cDNA fragments for taproot expressed genes. Isolation and molecular analysis of six cDNAs encoding the complete gene product revealed that these genes comprise homologues of a drought-inducible linker histone, a homologue of a major latex-like protein, a phosphoenolpyruvate carboxylase kinase, a putative vacuolar processing enzyme, a thaumatin-like protein and an alanine- and glutamic acid-rich protein. All genes are transcribed in taproots while transcription in leaves is low or undetectable.