These authors contributed equally to this work Summary• Certain plant species hyperaccumulate selenium (Se) up to 0.6% of their dry weight. It is not known whether Se hyperaccumulation offers the plants any advantage. In this study the hypothesis was tested that Se can protect plants from invertebrate herbivory or fungal infection.• Indian mustard ( Brassica juncea ) plants grown with or without Se were subjected to herbivory by caterpillars ( Pieris rapae ) and snails ( Mesodon ferrissi ), or to fungal infection by a root /stem pathogen ( Fusarium sp.) and a leaf pathogen ( Alternaria brassicicola ).• When given a choice between leaves with or without Se (0.1% Se of leaf d. wt), the caterpillars strongly preferred leaves without Se ( P < 0.01), while the snails preferred leaves containing Se ( P < 0.015). When consumed, the Se leaves were lethal to the caterpillars. The snails showed no toxicity symptoms, even though their tissue Se concentrations were comparable with the caterpillars. Se-containing plants were less susceptible to infection by both fungi.• In conclusion, Se was shown to protect Indian mustard plants from fungal infection and from herbivory by caterpillars, but not by snails.

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5Ј phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.NifS-like proteins are pyridoxal 5Ј phosphate (PLP)-dependent enzymes with sequence similarity to the Cys desulfurase encoded by nifS of Azotobacter vinelandii (Zheng et al., 1993). These proteins have been found in most organisms tested, where they play a role in S or Se metabolism (Mihara et al., 1997). NifS-like proteins catalyze the breakdown of Cys to form Ala and elemental S, or they may act on related substrates such as seleno-Cys to form Ala and elemental Se (Mihara et al., 1997). The nifS of A. vinelandii is required under nitrogen fixation conditions for the formation of Fe-S clusters in nitrogenase (Zheng et al., 1993). A. vinelandii NIFS is present in a gene cluster with several other genes (nifU, nifA, and cysE) all thought to be involved in Fe-S cluster formation. A second NifS-like protein of A. vinelandii, IscS, has a housekeeping function in the formation of other cellular Fe-S proteins (Zheng et al., 1993). Interestingly, iscS is present in a gene cluster that contains paralogs of the nif genes (iscU and iscA), thus, the nif and isc clusters share a similar organization (Zheng et al., 1998). Homologs of the nif/isc genes, all thought to play a role in cellular Fe-S cluster formation have been discovered in several other bacteria including in Escherichia coli (Zheng et al., 1998). In the eukaryotes, Fe-S clusters are essential cofactors for mitochondrial respiration, as well as for many cytosolic proteins. Recent work has suggested that in yeast and in mammals, all Fe-S clusters are made in the mitochondria (for review, see Lill and Kispal, 2000). Fe-S cluster formation in the mitochondria of eukaryotes involves homologs of the genes encoded by the nif/isc clusters of bacteria (Kispal et a...

We report here the characterization of Tri10, a novel regulatory gene within the trichothecene gene cluster. Comparison of Tri10 genomic and mRNA sequences revealed that removal of a single 77-bp intron provided a 1,260-bp open reading frame, encoding a 420-amino-acid protein. Disruption of Tri10 in Fusarium sporotrichioides abolished T-2 toxin production and dramatically decreased the transcript accumulation for four trichothecene genes (Tri4, Tri5, Tri6, and Tri101) and an apparent farnesyl pyrophosphate synthetase (Fpps) gene. Conversely, homologous integration of a disruption vector by a single upstream crossover event significantly increased T-2 toxin production and elevated the transcript accumulation of the trichothecene genes and Fpps. Further analysis revealed that disruption of Tri10, and to a greater extent the disruption of Tri6, increased sensitivity to T-2 toxin under certain growth conditions. Although Tri10 is conserved in Fusarium graminearum and Fusarium sambucinum and clearly plays a central role in regulating trichothecene gene expression, it does not show any significant matches to proteins of known or predicted function or to motifs except a single transmembrane domain. We suggest a model in which Tri10 acts upstream of the clusterencoded transcription factor TRI6 and is necessary for full expression of both the other trichothecene genes and the genes for the primary metabolic pathway that precedes the trichothecene biosynthetic pathway, as well as for wild-type levels of trichothecene self-protection. We further suggest the presence of a regulatory loop where Tri6 is not required for the transcription of Tri10 but is required to limit the expression of Tri10.The trichothecenes represent a large family of toxic secondary metabolites produced by a variety of filamentous fungi, including Fusarium, Myrothecium, Stachybotrys, Trichoderma, and Trichothecium (16). They are primarily found as contaminants in food and animal feed, and consumption of these compounds by humans or livestock results in vomiting, alimentary hemorrhaging, and dermatitis (20). These toxins are potent inhibitors of eukaryotic protein synthesis (23) and induce apoptosis (24). In plants the trichothecenes are also phytotoxic and have been associated with virulence in specific plantpathogen interactions (8,9,12,25).

Filamentous fungi elaborate a complex array of secondary metabolites, including antibiotics and mycotoxins. As many of these compounds pose significant economic and health concerns, elucidation of the underlying cellular mechanisms that control their production is essential. Previous work revealed that synthesis of the carcinogenic mycotoxins sterigmatocystin (ST) and aflatoxin (AF) in Aspergillus species is negatively controlled by FadA, the α‐subunit of a heterotrimeric G‐protein. In sharp contrast, we show here that the dominant activating fadA allele, fadAG42R, stimulates transcription of a gene from the A. nidulans penicillin (PN) gene cluster and elevates penicillin production. Thus, FadA has opposite roles in regulating the biosynthesis of a potent antibiotic (PN) and a lethal mycotoxin (ST) in A. nidulans. Furthermore, expression of fadAG42R in Fusarium sporotrichioides increases trichothecene (TR) mycotoxin production and alters TR gene expression. Our findings reveal that a G‐protein defines an important control point for differential expression of fungal secondary metabolites within and across fungal genera. These data provide critical evidence suggesting that targeting G‐protein signal transduction pathways as a means of controlling or preventing the production of a single mycotoxin could have serious undesirable consequences with regard to the production of other secondary metabolites.

Selenium (Se) is an essential element for many organisms but is toxic at higher levels. CpNifS is a chloroplastic NifS-like protein in Arabidopsis (Arabidopsis thaliana) that can catalyze the conversion of cysteine into alanine and elemental sulfur (S 0 ) and of selenocysteine into alanine and elemental Se (Se 0 ). We overexpressed CpNifS to investigate the effects on Se metabolism in plants. CpNifS overexpression significantly enhanced selenate tolerance (1.9-fold) and Se accumulation (2.2-fold). CpNifS overexpressors showed significantly reduced Se incorporation into protein, which may explain their higher Se tolerance. Also, sulfur accumulation was enhanced by approximately 30% in CpNifS overexpressors, both on media with and without selenate. Root transcriptome changes in response to selenate mimicked the effects observed under sulfur starvation. There were only a few transcriptome differences between CpNifS-overexpressing plants and wild type, besides the 25-to 40-fold increase in CpNifS levels. Judged from x-ray analysis of near edge spectrum, both CpNifS overexpressors and wild type accumulated mostly selenate (Se VI ). In conclusion, overexpression of this plant NifS-like protein had a pronounced effect on plant Se metabolism. The observed enhanced Se accumulation and tolerance of CpNifS overexpressors show promise for use in phytoremediation.

Tri10, a regulatory gene in trichothecene mycotoxin-producing Fusarium species, is required for trichothecene biosynthesis and the coordinated expression of four trichothecene pathway-specific genes (Tri4, Tri5, Tri6, and Tri101) and the isoprenoid biosynthetic gene for farnesyl pyrophosphate synthetase (FPPS). We showed that six more trichothecene genes (Tri3, Tri7, Tri8, Tri9, Tri11, and Tri12) are regulated by Tri10. We also constructed a cDNA library from a strain of Fusarium sporotrichioides that overexpresses Tri10 (1Tri10) and used cDNA derived from the 1Tri10 strain and a non-Tri10-expressing strain (⌬Tri10) to differentially screen macroarrays prepared from the cDNA library. This screen identified 15 additional Tri10-regulated transcripts. Four of these transcripts represent Tri1, Tri13, and Tri14 and a gene designated Tri15. Three other sequences are putative orthologs of genes for isoprenoid biosynthesis, the primary metabolic pathway preceding trichothecene biosynthesis. The remaining eight sequences have been designated Ibt (influenced by Tri10) genes. Of the 26 transcripts now known to be positively regulated by Tri10, 22 are positively coregulated by Tri6, a gene that encodes a previously characterized trichothecene pathway-specific transcription factor. These 22 Tri10-and Tri6-coregulated sequences include all of the known Tri genes (except for Tri10), the FPPS gene, and the other three putative isoprenoid biosynthetic genes. Tri6 also regulates a transcript that is not regulated by Tri10. Thus, Tri10 and Tri6 regulate overlapping sets of genes that include a common group of multiple genes for both primary and secondary metabolism.

The chloroplast contains many iron (Fe)-sulfur (S) proteins for the processes of photosynthesis and nitrogen and S assimilation. Although isolated chloroplasts are known to be able to synthesize their own Fe-S clusters, the machinery involved is largely unknown. Recently, a cysteine desulfurase was reported in Arabidopsis (Arabidopsis thaliana; AtCpNifS) that likely provides the S for Fe-S clusters. Here, we describe an additional putative component of the plastid Fe-S cluster assembly machinery in Arabidopsis: CpIscA, which has homology to bacterial IscA and SufA proteins that have a scaffold function during Fe-S cluster formation. CpIscA mRNA was shown to be expressed in all tissues tested, with higher expression level in green, photosynthetic tissues. The plastid localization of CpIscA was confirmed by green fluorescent protein fusions, in vitro import, and immunoblotting experiments. CpIscA was cloned and purified after expression in Escherichia coli. Addition of CpIscA significantly enhanced CpNifS-mediated in vitro reconstitution of the 2Fe-2S cluster in apo-ferredoxin. During incubation with CpNifS in a reconstitution mix, CpIscA was shown to acquire a transient Fe-S cluster. The Fe-S cluster could subsequently be transferred by CpIscA to apo-ferredoxin. We propose that the CpIscA protein serves as a scaffold in chloroplast Fe-S cluster assembly.

Plastids are known to be able to synthesize their own iron-sulfur clusters, but the biochemical machinery responsible for this process is not known. In this study it is investigated whether CpNifS, the chloroplastic NifS-like cysteine desulfurase of Arabidopsis thaliana (L.) Heynh. is responsible for the release of sulfur from cysteine for the biogenesis of iron-sulfur (Fe-S) clusters in chloroplasts. Using an in vitro reconstitution assay it was found that purified CpNifS was sufficient for Fe-S cluster formation in ferredoxin in the presence of cysteine and a ferrous iron salt. Antibody-depletion experiments using stromal extract showed that CpNifS is also essential for the Fe-S cluster formation activity of chloroplast stroma. The activity of CpNifS in the stroma was 50- to 80-fold higher than that of purified CpNifS on a per-protein basis, indicating that other stromal factors cooperate in Fe-S cluster formation. When stromal extract was separated on a gel-filtration column, most of the CpNifS eluted as a dimer of 86 kDa, but a minor fraction of the stromal CpNifS eluted at a molecular weight of approx. 600 kDa, suggesting the presence of a multi-protein complex. The possible nature of the interacting proteins is discussed.