niiA Genes Research Articles

Polarity-Defective Mutants of Aspergillus nidulans

We have identified two polarity-defective (pod) mutants in Aspergillus nidulans from a collection of heat-sensitive lethal mutants. At restrictive temperature, these mutants are capable of nuclear division but are unable to establish polar hyphal growth. We cloned the two pod genes by complementation of their heat-sensitive lethal phenotypes. The libraries used to clone the pod genes are under the control of the bidirectional niaD and niiA promoters. Complementation of the pod mutants is dependent on growth on inducing medium. We show that rescue of the heat-sensitive phenotype on inducing media is independent of the orientation of the gene relative to the niaD or niiA promoters, demonstrating that the intergenic region between the niaD and the niiA genes functions as an orientation-independent enhancer and repressor that is capable of functioning over long distances. The products of the podG and the podH genes were identified as homologues of the α subunit of yeast mitochondrial phenylalanyl–tRNA synthetase and transcription factor IIF interacting component of the CTD phosphatase. Neither of these gene products would have been predicted to produce a pod mutant phenotype based on studies of cellular polarity mutants in other organisms. The implications of these results are discussed.

Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing.

Homology-dependent gene silencing is a regulatory mechanism that limits RNA accumulation from affected loci either by suppression of transcription (transcriptional gene silencing, TGS) or by activation of a sequence-specific RNA degradation process (post-transcriptional gene silencing, PTGS). The P1/HC-Pro sequence of plant potyviruses and the 2b gene of the cucumber mosaic virus have been shown to interfere with PTGS. The ability of these viral suppressors of PTGS to interfere with TGS was tested using the 271 locus which imposes TGS on transgenes under 35S or 19S promoters and PTGS on the endogenous nitrite reductase gene (Nii). Both P1/HC-Pro and 2b reversed PTGS of Nii genes in 271-containing tobacco plants, but failed to reverse TGS of 35S-GUS transgenes in the same plant. P1/HC-Pro expression from a transgene also failed to suppress either the initiation or maintenance of TGS imposed by the NOSpro-silencing locus, H2. These results indicate that PTGS and TGS operate through unlinked pathways or that P1/HC-Pro and 2b interfere at step(s) in PTGS that are downstream of any common components in the two pathways. The data suggest a simple assay to identify post-transcriptionally silenced transgenic lines with the potential to be stably converted to high expressing lines.

Expression of a yeast RNase III gene in transgenic tobacco silences host nitrite reductase genes.

When a gene encoding the Schizosaccharomyces pombe dsRNA-specific RNase III, pac1, was expressed in transgenic tobacco plants, six out of thirteen transformed plants gave progeny among which were individuals displaying a distinctive chlorotic phenotype. These chlorotic plants strongly resemble those transformed with a 35S-Nii (nitrite reductase) transgene, in which both Nii host genes and the 35S-Nii transgene are silenced by co-suppression. RNA blots showed that the host Nii genes were silenced in chlorotic 35S-pac1 plants but not in individuals with a normal green phenotype. Neither the transcript levels of the other cellular genes tested nor the transcription of Nii genes was significantly affected by the expression of pac1. This is the first observation of post-transcriptional silencing of host genes by a transgene with no apparent sequence similarity to the target gene.

Expression and sequence requirements for nitrite reductase co-suppression.

We have previously reported that the introduction of a full-length tobacco nitrite reductase Nii1 cDNA under the control of the 35S promoter triggers co-suppression of endogenous Nii genes in 25% of tobacco transformants. Here we show that introduction of chimeric Nii1-uidA, uidA-Nii1 and Nii1-uidA-Nii1 transgenes carrying 186 bp of the 5' end and/or 241 bp of the 3' end of the Nii1 cDNA do not trigger co-suppression of endogenous Nii genes. In addition, we show that when introduced by crossing or transformation into co-suppressed transgenic tobacco lines carrying full-length Nii1 transgenes, these chimeric transgenes are not silenced. These results therefore suggest that the 5' and 3' ends of the Nii1 cDNA are not sufficient to trigger co-suppression and are not targets for homology-dependent RNA degradation. Surprisingly, co-suppression was released in a double transformant obtained by introduction of one of these constructs into the co-suppressed transgenic tobacco line 461-2.1 homozygous for a full-length Nii1 transgene, and in one plant regenerated from untransformed leaf discs (plant 461-2.1*). The reappearance of co-suppression at very low frequency (less than 10(-3)) in the F2 progeny of plant 461-2.1* and the apparent absence of structural modification of the transgene locus suggest a metastable epigenetic modification. The steady-state level of Nii mRNAs in the plant 461-2-.1* was higher than in wild-type plants but lower than in hemizygous plants 461-2.1 which never trigger silencing. These results therefore confirm that transcription of the transgene above a particular threshold is required to trigger co-suppression.

Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions.

Using grafting procedures, we investigated the transmission of co-suppression of nitrate reductase and nitrite reductase host genes and transgenes and of post-transcriptional silencing of a uidA transgene encoding glucuronidase in tobacco. We demonstrate that silencing is transmitted with 100% efficiency from silenced stocks to non-silenced scions expressing the corresponding transgene. Transmission is unidirectional from stock to scion, transgene specific, locus independent and requires the presence of a transcriptionally active transgene in the target scion. The transmission of co-suppression occurs when silenced stocks and non-silenced target scions are physically separated by up to 30 cm of stem of a non-target wild-type plant. Taken together, these results suggest that a non-metabolic, transgene-specific, diffusable messenger mediates the propagation of de novo post-transcriptional silencing through the plant.

Nitrate reductase and nitrite reductase as targets to study gene silencing phenomena in transgenic plants

Nitrate assimilation is a fundamental function in any plant including those that can fix atmospheric nitrogen in symbiosis with soil bacteria. In recent years, attempts have been made to understand the biological significance of the complex regulation of this pathway using genetic engineering techniques. Transgenic plants that either over- or under-express genes of the nitrate assimilation pathway were created in order to determine whether such directed changes affect the regulation of the metabolism. Apart from interesting physiological results, unexpected gene silencing phenomena have been observed resulting from the introduction of five different transgenes derived from either the tobacco Nia or Nii genes encoding nitrate reductase and nitrite reductase, respectively. In this review, each of these five silencing phenomena is described, with the emphasis on the advantages provided by the use of both Nia and Nii genes to analyze the molecular and genetic basis of gene silencing in transgenic plants.

Sequence homology requirements for transcriptional silencing of 35S transgenes and post-transcriptional silencing of nitrite reductase (trans)genes by the tobacco 271 locus.

The transgene locus of the tobacco plant 271 (271 locus) is located on a telomere and consists of multiple copies of a plasmid carrying an NptII marker gene driven by the cauliflower mosaic virus (CaMV) 19S promoter and the leaf-specific nitrite reductase Nii1 cDNA cloned in the antisense orientation under the control of the CaMV 35S promoter. Previous analysis of gene expression in leaves has shown that this locus triggers both post-transcriptional silencing of the host leaf-specific Nii genes and transcriptional silencing of transgenes driven by the 19S or 35S promoter irrespective of their coding sequence and of their location in the genome. In this paper we show that silencing of transgenes carrying Nii1 sequences occurs irrespective of the promoter driving their expression and of their location within the genome. This phenomenon occurs in roots as well as in leaves although root Nii genes share only 84% identity with leaf-specific Nii1 sequences carried by the 271 locus. Conversely, transgenes carrying the bean Nii gene (which shares 76% identity with the tobacco Nii1 gene) escape silencing by the 271 locus. We also show that transgenes driven by the figwort mosaic virus 34S promoter (which shares 63% identity with the 35S promoter) also escape silencing by the 271 locus. Taken together, these results indicate that a high degree of sequence similarity is required between the sequences of the silencing locus and of the target (trans)genes for both transcriptional and post-transcriptional silencing.

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster.

The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.

Use of the biolistic process for the analysis of nitrate-inducible promoters in transient expression assays

The expression of reporter genes under the control of nitrate reductase or nitrite reductase promoters measured in bombarded leaf tissues was compared to transcript levels expressed from the resident Nia gene or to that of a reporter gene under the control of nitrite reductase promoter stably inserted into the nuclear genome. It was found that transient assays do not reflect the expression of the genomic integrated sequences. Particularly striking was the absence of repression of the reporter genes by ammonium or glutamine in transient assays. The limits of the use of particle bombardment in transient experiments are discussed. One of the different hypotheses explaining the data involved a model of a negative control mediating the repression of Nia and Nii genes expression on reduced nitrogen source.

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5'CTCCGHGG3'. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.

The use of mutants and transgenic plants to study nitrate assimilation

ABSTRACTThe nitrate assimilatory pathway has been the matter of intensive genetic and molecular analysis over the past decade. Mutants impaired in the expression of nitrate reductase have been characterized in a number of plant species. Molecular analysis of the Nia gene coding for nitrate reductase has been the basis for a three‐domain model of the structure of the enzyme, in agreement with biochemical and genetic data. Mutagenesis and antisense strategies have led to the description of nitrite reductase deficiencies. The molecular analysis of the corresponding Nii genes has provided invaluable information on the structure of nitrite reductase. Recently, a gene involved in nitrate uptake has also been identified. The regulation of the nitrate assimilatory pathway has been investigated. Analysis of the regulation of the pathway at the molecular level has shown evidence for the involvement of nitrate, light and/or sucrose, and reduced nitrogen in the regulation. Surprisingly, no bona fide regulatory mutant specific to this pathway has been identified so far in higher plants. This may reflect the redundancy of regulatory genes. The deregulated expression of one or the other step of the pathway obtained by ectopic expression of the corresponding genes is a new approach to study the physiological role of these regulations. Elements of the pathway have also been successfully used as transposon traps, or negatively selectable markers for other purposes. Finally, the identification at the molecular level of regulatory genes and structural elements involved in transport and storage of nitrate, or in the biosynthesis of cofactors of nitrate and nitrite reductases, will be the goal of the next decade.

The Aspergillus niger niaD gene encoding nitrate reductase: upstream nucleotide and amino acid sequence comparisons

The Aspergillus niger niaD gene has been sequenced and the inferred nitrate reductase (NR) protein found to consist of 867 amino acid residues (97 kDa). The gene is interrupted by six small introns, as deduced by comparison with the niaD gene of Aspergillus nidulans. The positions of these putative introns are conserved between the two fungi, although the sequences are dissimilar. The niiA gene, encoding nitrite reductase, the second reductive step in the nitrate assimilation pathway, is tightly linked to niaD and divergently transcribed in A. niger, similar to the general organisation in the related fungi, Aspergillus oryzae and A. nidulans. The nucleotide (nt) sequences of the intergenic region between niiA and niaD (excluding the ATG translation start codon) of A. niger (1668 nt) and A. oryzae (1575 nt) were determined and compared with the previously determined A. nidulans (1262 nt) sequence. No striking extended nt regions of homology are observed in spite of the fact that transgenic strains with fungal niaD or the two control genes required for induction and repression show virtually normal regulation. Fungal NR shows considerable aa homology with higher plant NR, particularly within the co-factor domains for flavin adenoside dinucleotide, heme and molybdopterin cofactor.

Cloning of the nitrate - nitrite reductase gene cluster of Penicillium chrysogenum and use of the niaD gene as a homologous selection marker

A new homologous transformation system for the filamentous fungus Penicillium chrysogenum is described. The system is based on complementation of niaD mutants using the nitrate reductase structural gene ( niaD of P. chrysogenum. Spontaneous niaD mutants were identified after selection for chlorate resistance, in growth tests and subsequent complementation with the niaD gene of Aspergillus oryzae. The P. chrysogenum niaD gene was isolated from a genomic library using the Aspergillus nidulans niaD gene as a probe. After subcloning of the hybridizing fragment, the vector obtained, pPC1-1, was capable of transforming a P. chrysogenum niaD mutant at an average of 40 transformants per μg of circular DNA. Southern analysis of genomic DNA from a number of transformants showed that pPC1-1 DNA was integrated predominantly at sites other than the niaD locus. Using hybridization analysis it was shown that the niaD gene of P. chrysogenum is clustered with the nitrite reductase gene ( niiA). From analysis of the nucleotide sequences of parts of the niaD and niiA genes of P. chrysogenum and comparison of these sequences with nucleotide sequences of the corresponding A. nidulans genes it was deduced that the P. chrysogenum genes are divergently transcribed.

GENETIC STUDIES OF NITRATE ASSIMILATION IN ASPERGILLUS NIDULANS

Summary(1) In Aspergillus nidulans, at least 16 genes can mutate to affect the reduction of nitrate to ammonium, a process requiring two enzymes, nitrate reductase and nitrite reductase.(2) niaD is the only gene whose effects on enzyme structure are confined to nitrate reductase alone. It specifies a core polypeptide, one or more of which form the basic subunit of nitrate reductase, molecular weight 50000.(3) At least five cnx genes together specify a molybdenum co‐factor, necessary for the activity of nitrate reductase, and of xanthine dehydrogenases I and II. The cnxH gene specifies a polypeptide component of this co‐factor, and the cnxE and F gene products are involved in co‐factor elaboration, The role of the remaining cnx genes is at present unknown.(4) Functional nitrate reductase has a molecular weight of 200000 and is likely to consist of four subunits, together with one or more molecules of the cnx‐specified co‐factor.(5) The co‐factor plays a catalytic role in the aggregation of nitrate‐reductase subunits.(6) The niiA gene is the structural gene for nitrite reductase.(7) Other genes affecting nitrate assimilation are either regulatory or bring about their effects indirectly.(8) Of the genes affecting nitrate assimilation, close linkage is found only between the niiA and niaD genes.(9) Nitrate and nitrite reductases are subject to control by nitrate induction and ammonium repression.(10) Nitrate induction is mediated by the nirA gene whose product must be active for the niiA and niaD genes to be expressed. Since most niaD mutants produce nitrite reductase constitutively, it is likely that the nirA gene product is normally inactivated by nitrate reductase, but only when the latter is not complexed with nitrate,(11) Ammonium repression is mediated by the areA gene, whose product must be active for the expression of the niiA and niaD genes, and which is inactive in the presence of ammonium.(12) The tamA gene may function similarly to the areA gene, both gene products being necessary for the expression of the niiA and niaD genes.(13) Although the niiA and niiD genes are probably contiguous, they are not likely to be organized into a structure equivalent to a bacterial operon.(14) Whereas the areA and nirA genes regulate the synthesis of nitrate and nitrite reductases, it is probable that at least nitrate reductase is also subject to post‐translational control, the presence of active enzyme being correlated with high levels of NADPH.(15) The regulation of the pentose‐phosphate pathway, of mannitol‐I‐phosphate dehydrogenase and of certain activities required for the catabolism of some nitrogen‐containing compounds appears to be connected with that of nitrate assimilation. In all cases, it is probable that the nirA gene and nitrate reductase itself are involved.

Cholorate toxicity in Aspergillus nidulans: the selection and characterisation of chlorate resistant mutants.

Mutation in at least ten genes can result in chlorate reistance in Aspergillus nidulans. Mutation in seven of these genes also results in the inability to use nitrate as nitrogen source. The various classes of resistant mutant obtained occur in different proportions, depending on whether or not a mutagenic treatment is employed, and also on which nitrogen source is used for selection. The prinicipal effect of mutagen arises because mutations in the niaD gene, the nitrate reductase structural gene, are relatively much commoner when no mutagen is used than after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. This may be connected with the finding that deletions involving the niaD gene are relatively more common among samples of spontaneous niaD mutants. Some of these deletions extend to the neighbouring niiA gene, the structural gene for nitrite reductase. The selection procedures used were designed to avoid bias in favour of any particular chlorate resistant phenotype. Even if biases existed however, these could not account for the variation found from nitrogren source to nitrogen source in the proportions of certain resistant classes having apparently identical chlorate resistance phenotypes.