Kenwyn R. Gayler

Role of RNA and protein synthesis in stimulated germination of zoospores of the pathogenic fungusPhytophthora palmivora

Abstract The role of RNA and protein synthesis in differentiating spores of the fungus Phytophthora palmivora was investigated using inhibitors, precursor assimilation, and in vitro translation of directly extracted RNA. Distinct differences in the capacity to take up small molecules, including exogenous precursors of protein and RNA synthesis, were detected between the different stages of differentiation. Zoospores and cysts of this fungus were impermeable to amino acids, glucose, and inorganic phosphate. Uptake of these metabolites was observed only after emergence of the germ tube. By contrast, inhibitors of protein and RNA synthesis penetrated these cells. In the presence of either cycloheximide or actinomycin D, the cells remained as cysts and did not germinate. Although encystment was not dependent on de novo transcription or translation, an increase in the pool of polyadenylated RNA was detected in the cells during this stage. This polyadenylated RNA showed a rapid and sustained increase in template activity, as measured by cell-free translation assays. The increase commenced very early in the cyst stage, immediately following pectin stimulation, and was attributed to the transcription of new mRNA. This transcription during encystment, which was also shown to be inhibited by actinomycin D, appears to be essential for germination in this species of fungus.

The Structure of Ap4A Hydrolase Complexed with ATP-MgFx Reveals the Basis of Substrate Binding

Ap(4)A hydrolases are Nudix enzymes that regulate intracellular dinucleoside polyphosphate concentrations, implicating them in a range of biological events, including heat shock and metabolic stress. We have demonstrated that ATP x MgF(x) can be used to mimic substrates in the binding site of Ap(4)A hydrolase from Lupinus angustifolius and that, unlike previous substrate analogs, it is in slow exchange with the enzyme. The three-dimensional structure of the enzyme complexed with ATP x MgF(x) was solved and shows significant conformational changes. The substrate binding site of L. angustifolius Ap(4)A hydrolase differs markedly from the two previously published Nudix enzymes, ADP-ribose pyrophosphatase and MutT, despite their common fold and the conservation of active site residues. The majority of residues involved in substrate binding are conserved in asymmetrical Ap(4)A hydrolases from pathogenic bacteria, but are absent in their human counterparts, suggesting that it might be possible to generate compounds that target bacterial, but not human, Ap(4)A hydrolases.

Biosynthesis, cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius.

The biosynthesis of conglutin δ has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin δ formed the major sink for [35S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin δ was determined. The structure of the precursor polypeptide for conglutin δ predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of Mr 4520, together with a linking region of 13 amino acids and a subunit polypeptide of Mr 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin δ. Comparison of the sequences of conglutin δ with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin δ, were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn2+- or Mg2+-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.

Elicitins: proteins in search of a role?

The literature on elicitins, proteins secreted by many species of Phytophthora and some species of the related genus Pythium in culture, is reviewed. The review covers both the properties of elicitins as proteins and their role in the biology of the secreting organism. It is proposed that in plant species other than tobacco, elicitins play no role in the determination of pathogenicity. In the specific case of tobacco they appear to have taken on this role, possibly as a result of mutation in a tobacco cell membrane protein associated with some aspect of transmembrane signalling. It is suggested that the altered affinity of elicitins for this mutant plant protein resulted in a change in the regulation of the signal transduction pathway, inducing a hypersensitive-like response in the presence of elicitins, resulting in a new role for these proteins as avirulence factors.

A Comprehensive Bioinformatics Analysis of the Nudix Superfamily in Arabidopsis thaliana

Nudix enzymes are a superfamily with a conserved common reaction mechanism that provides the capacity for the hydrolysis of a broad spectrum of metabolites. We used hidden Markov models based on Nudix sequences from the PFAM and PROSITE databases to identify Nudix hydrolases encoded by the Arabidopsis genome. 25 Nudix hydrolases were identified and classified into 11 individual families by pairwise sequence alignments. Intron phases were strikingly conserved in each family. Phylogenetic analysis showed that all multimember families formed monophyletic clusters. Conserved familial sequence motifs were identified with the MEME motif analysis algorithm. One motif (motif 4) was found in three diverse families. All proteins containing motif 4 demonstrated a degree of preference for substrates containing an ADP moiety. We conclude that HMM model-based genome scanning and MEME motif analysis, respectively, can significantly improve the identification and assignment of function of new members of this mechanistically-diverse protein superfamily.

Structure of the cDNA coding for conglutin γ, a sulphur-rich protein from Lupinus angustifolius

The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin γ, was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of Mr 28 239 and 16 517. Extensive sequence homology between the protein encoded by conglutin γ cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin γ. It is concluded that conglutin γ is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.

Detection and characterization of a new β-conglycinin from soybean seeds

Abstract A new protein has been isolated from the reserve proteins of the seeds of soybean ( Glycine max ) which is particularly deficient in methionine and cysteine. The protein dissociated in sodium dodecyl sulfate into a single polypeptide, M r 48,000. The amino acid composition, N-terminal leucine and mobility on gel electrophoresis of this polypeptide all were indistinguishable from the β-subunit of β-conglycinin. In its nondissociated form, the protein behaved as a trimer of M r , 137,000 ± 4000. Its sedimentation coefficient at ionic strength 0.5 was 7.5 S and it possessed antigenic determinants in common with β-conglycinin. This protein therefore has the properties of a new isomer of β-conglycinin—a homogeneous trimer of β subunits.

Transcription of genes for conglutin γ and a leginsulin-like protein in narrow-leafed lupin

The expression of genes encoding conglutin γ and a leginsulin-like protein has been examined in narrow-leafed lupin, Lupinus angustifolius L. Conglutin γ is a homologue of basic 7S globulin (Bg), the insulin and leginsulin binding protein from soybean. Accumulation of conglutin γ mRNA, as assessed by northern assays and reverse-transcription PCR, was tightly regulated both spatially and temporally in lupin plants and was detected almost exclusively in developing seeds. Similar tissue and temporal specificity was demonstrated when 1.8 kb of the promoter region from the conglutin γ gene was used to drive the expression of a β-glucuronidase reporter gene in transgenic plants. In stably transformed tobacco the conglutin γ promoter produced strong, temporally regulated and seed-specific expression of the reporter gene which was localised to the embryo tissues and to a layer of cells adjacent to the seed coat. A truncated 0.29 kb promoter fragment produced much reduced levels of expression and a loss of embryo specificity. Leginsulin-like mRNA was similarly detected in lupins only in developing seeds. The leginsulin-like gene detected in L. angustifolius showed 96% sequence identity to leginsulin from soybean within the 280 bp region amplified from lupin by PCR. The results demonstrate that both components of a Bg-leginsulin putative signal transduction pathway are present in the seeds of lupin.

Interactions between elicitins and radish Raphanus sativus

Abstract. Two responses to elicitins are described in cultivars of radish (Raphanus sativus L.). Type I, exhibited by the cultivar Daikon, is characterised by wilting and desiccation within 24 h of elicitin application and was previously reported as the sensitive response (S. Kamoun et al. 1993, Mol Plant-Microbe Interact 6: 15–25). At 1 μg elicitin · g−1 FW radish tissue, symptoms appeared after 8 h, a sensitivity comparable to that shown by tobacco to β elicitins (J.-C. Pernollet et al., 1993, Physiol Mol Plant Pathol 42: 53–67; S. Kamoun et al., 1993, Mol Plant-Microbe Interact 6: 15–25). Elicitin failed to induce these symptoms in the cultivar White Icicle, even at 100 μg · g−1 FW of tissue. However, a different response (Type II) with symptoms resembling senescence appeared in White Icicle after 48 h and were fully developed by 72 h. The Type II response was induced at levels of elicitin above 0.3 μg · g−1 FW. Elicitin-treated Daikon leaves held at 100% relative humidity, rather than ambient (50–60%) did not wilt and by 72 h displayed Type II symptoms. When treated Daikon leaves were removed to ambient humidity at any time during the latent period, they developed Type I symptoms within 2 h. Although Type I symptoms were suppressed in Daikon at high humidity, there was no indication that leaf diffusion resistance or plant water conductance were affected. Protoplasts from the cultivar Daikon responded to elicitin by H+ uptake and K+ release, with maximal response at 300 pM. The response was eliminated by K252a or staurosporine. Daikon protoplasts also showed transient uptake/secretion of Ca2+ on elicitin addition. Protoplasts from White Icicle gave neither of these responses. Both Daikon and White Icicle phenotypes could be transferred to progeny of Daikon-White Icicle crosses and in the F2 generation three phenotypes, including a null, segregated. Only those F2 plants which exhibited the Daikon phenotype produced protoplasts which responded to elicitin.