Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.     

<Emphasis Type="Italic">Streptomyces scabiei</Emphasis> and its toxin thaxtomin A induce scopoletin biosynthesis in tobacco and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Streptomyces scabiei is the predominant causal agent of common scab of potato in North America. The virulence of common scab-causing streptomycetes relies on their capacity to synthesize thaxtomins. In this study, the effects of S. scabiei infection and of thaxtomin A, the main toxin produced by S. scabiei, were tested for the elicitation of plant defense molecules in the model plants tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Tobacco leaves infected with spores of S. scabiei strain EF-35 or infiltrated with purified thaxtomin A produced a blue fluorescent compound that was not detected in leaves infiltrated with spores of a S. scabiei mutant deficient in thaxtomin A biosynthesis. Thin layer chromatography and high performance liquid chromatography identified this fluorescent compound as scopoletin, a plant defense phytoalexin. Arabidopsis seedlings grown in liquid medium also excreted scopoletin as a reaction to S. scabiei and thaxtomin A. The effects of the presence of scopoletin on S. scabiei were also investigated. The phytoalexin scopoletin caused a slight reduction of bacterial growth and a severe decrease of thaxtomin A production. Scopoletin was shown to inhibit thaxtomin A production by repression of a gene involved in the toxin biosynthesis.     

Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.     

Plant cell growth and ion flux responses to the streptomycete phytotoxin thaxtomin A: calcium and hydrogen flux patterns revealed by the non-invasive MIFE technique

Thaxtomin A, a key phytotoxin produced by plant pathogenic Streptomyces sp., is implicit in common scab disease expression in potato. Primary targets and modes of action of thaxtomin A toxicity in plant cells are not well understood. In this work, early signalling events associated with thaxtomin A toxicity were studied using the ion-selective microelectrode ion flux estimation (MIFE) technique. Thaxtomin A-induced changes in net ion fluxes were measured across the plasma membrane (PM) of root and pollen tube tissue in Arabidopsis thaliana and tomato. Within a minute after toxin application, a rapid and short-lived Ca2+ influx was observed. Well ahead of the marked inhibition of root growth, a significant shift towards net H+ efflux across the PM occurred in all tissues. Similar to root tissues, thaxtomin A significantly modified ion flux profiles from growing pollen tubes. Thaxtomin A was more effective in young, physiologically active tissues (root elongation zone or pollen tube apex), suggesting a higher density of thaxtomin A-binding sites in these regions. Overall, our data provide the first evidence that thaxtomin A triggers an early signalling cascade, which may be crucial in plant-pathogen interactions. It also suggests a possible interaction between thaxtomin A and PM auxin receptors, as revealed from experiments on the auxin-sensitive ucu2-2/gi2 A. thaliana mutant.     

Selection and Characterization of Microorganisms Utilizing Thaxtomin A, a Phytotoxin Produced by Streptomyces scabies

Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.     

The ability of Pseudomonas sp. LBUM 223 to produce phenazine-1-carboxylic acid affects the growth of Streptomyces scabies, the expression of thaxtomin biosynthesis genes and the biological control potential against common scab of potato

Streptomyces scabies causes common scab, an economical disease affecting potato crops world-wide, for which no effective control measure exists. This pathogen produces the plant toxin thaxtomin A, which is involved in symptom development on potato tubers. A biological control approach that can limit S. scabies growth and repress thaxtomin production represents an attractive alternative to classical control strategies. Pseudomonas sp. LBUM 223 produces phenazine-1-carboxylic acid (PCA), an antibiotic that inhibits the growth of plant pathogens and contributes to the biological control of plant diseases. In this study, the involvement of LBUM 223's PCA-producing ability in the growth inhibition of S. scabies, repression of thaxtomin biosynthesis genes (txtA and txtC) and the biological control of common scab of potato was investigated using a mutant defective in PCA production (LBUM 223phzC(-) ). Streptomyces scabies growth was inhibited to a significantly lesser degree by LBUM 223phzC(-) than by the wild type. LBUM 223 also significantly repressed txtA and txtC expression in S. scabies and protected potato against disease, whereas LBUM 223phzC(-) did not. These results suggest that PCA production is central to the ability of LBUM 223 to limit pathogen growth, repress the expression of key pathogenicity genes and control common scab of potato.     

Molecular and functional characterization of Arabidopsis Cullin 3A

Cullin proteins, which belong to multigenic families in all eukaryotes, associate with other proteins to form ubiquitin protein ligases (E3s) that target substrates for proteolysis by the 26S proteasome. Here, we present the molecular and genetic characterization of a plant Cullin3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CUL3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and is able to interact with the ring-finger protein RBX1. A genomic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families. Yeast two-hybrid experiments indicate that representative members of certain families are able to physically interact with both CUL3A and CUL3B, suggesting that Arabidopsis CUL3 forms E3 protein complexes with certain BTB domain proteins. In order to determine the function of CUL3A, we used a reverse genetic approach. The cul3a null mutant flowers slightly later than the control plants. Furthermore, this mutant exhibits a reduced sensitivity of the inhibition of hypocotyl growth in far-red light and miss-expresses COP1. The viability of the mutant plants suggests functional redundancy between the two CUL3 genes in Arabidopsis.     

The txtAB genes of the plant pathogen Streptomyces acidiscabies encode a peptide synthetase required for phytotoxin thaxtomin A production and pathogenicity

Four Streptomyces species have been described as the causal agents of scab disease, which affects economically important root and tuber crops worldwide. These species produce a family of cyclic dipeptides, the thaxtomins, which alone mimic disease symptomatology. Structural considerations suggest that thaxtomins are synthesized non-ribosomally. Degenerate oligonucleotide primers were used to amplify conserved portions of the acyladenylation module of peptide synthetase genes from genomic DNA of representatives of the four species. Pairwise Southern hybridizations identified a peptide synthetase acyladenylation module conserved among three species. The complete nucleotide sequences of two peptide synthetase genes (txtAB) were determined from S. acidiscabies 84.104 cosmid library clones. The organization of the deduced TxtA and TxtB peptide synthetase catalytic domains is consistent with the formation of N-methylated cyclic dipeptides such as thaxtomins. Based on high-performance liquid chromatography (HPLC) analysis, thaxtomin A production was abolished in txtA gene disruption mutants. Although the growth and morphological characteristics of the mutants were identical to those of the parent strain, txtA mutants were avirulent on potato tubers. Moreover, introduction of the thaxtomin synthetase cosmid into a txtA mutant restored both pathogenicity and thaxtomin A production, demonstrating a critical role for thaxtomins in pathogenesis.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.     

Effect of amino acids on thaxtomin A biosynthesis by Streptomyces scabies

The regulatory effect of amino acids on the production of thaxtomin A, a phytotoxin produced by Streptomyces scabies, was investigated. Tryptophan had an important inhibitory effect on the toxin biosynthesis in all five strains of S. scabies tested. Two other aromatic amino acids (tyrosine and phenylalanine) also inhibited thaxtomin A biosynthesis, while aliphatic amino acids did not cause an important decline in thaxtomin A production. Methylation of tryptophan prevented or reduced the inhibitory effect on thaxtomin A biosynthesis. In spite of the inhibitory action of tryptophan and phenylalanine on thaxtomin A production, incorporation of these radiolabeled molecules into thaxtomin A confirmed that they are metabolic precursors for the biosynthesis of the phytotoxin.     

TxtH is a key component of the thaxtomin biosynthetic machinery in the potato common scab pathogen Streptomyces scabies

Streptomyces scabies causes potato common scab disease, which reduces the quality and market value of affected tubers. The predominant pathogenicity determinant produced by S. scabies is the thaxtomin A phytotoxin, which is essential for common scab disease development. Production of thaxtomin A involves the nonribosomal peptide synthetases (NRPSs) TxtA and TxtB, both of which contain an adenylation (A-) domain for selecting and activating the appropriate amino acid during thaxtomin biosynthesis. The genome of S. scabies 87.22 contains three small MbtH-like protein (MLP)-coding genes, one of which (txtH) is present in the thaxtomin biosynthesis gene cluster. MLP family members are typically required for the proper folding of NRPS A-domains and/or stimulating their activities. This study investigated the importance of TxtH during thaxtomin biosynthesis in S. scabies. Biochemical studies showed that TxtH is required for promoting the soluble expression of both the TxtA and TxtB A-domains in Escherichia coli, and amino acid residues essential for this activity were identified. Deletion of txtH in S. scabies significantly reduced thaxtomin A production, and deletion of one of the two additional MLP homologues in S. scabies completely abolished production. Engineered expression of all three S. scabies MLPs could restore thaxtomin A production in a triple MLP-deficient strain, while engineered expression of MLPs from other Streptomyces spp. could not. Furthermore, the constructed MLP mutants were reduced in virulence compared to wild-type S. scabies. The results of our study confirm that TxtH plays a key role in thaxtomin A biosynthesis and plant pathogenicity in S. scabies.     

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N -methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.