An atypical forkhead‐containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)‐box‐containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)‐based gene silencing was employed to alter the expression of SsFkh1. RNA‐silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis‐related laccase genes and a polyketide synthase‐encoding gene were significantly down‐regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi‐silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.     

The PsbP Domain Protein 1 Functions in the Assembly of Lumenal Domains in Photosystem I

Photosystem I (PS I) is a multisubunit membrane protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase. The PsbP domain protein 1 (PPD1; At4g15510) is located in the thylakoid lumen of plant chloroplasts and is essential for photoautotrophy, functioning as a PS I assembly factor. In this work, RNAi was used to suppress PPD1 expression, yielding mutants displaying a range of phenotypes with respect to PS I accumulation and function. These PPD1 RNAi mutants showed a loss of assembled PS I that was correlated with loss of the PPD1 protein. In the most severely affected PPD1 RNAi lines, the accumulated PS I complexes exhibited defects in electron transfer from plastocyanin to the oxidized reaction center P₇₀₀⁺. The defects in PS I assembly in the PPD1 RNAi mutants also had secondary effects with respect to the association of light-harvesting antenna complexes to PS I. Because of the imbalance in photosystem function in the PPD1 RNAi mutants, light-harvesting complex II associated with and acted as an antenna for the PS I complexes. These results provide new evidence for the role of PPD1 in PS I biogenesis, particularly as a factor essential for proper assembly of the lumenal portion of the complex.     

A gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein export

A gene locus of Bacillus subtilis identified by mutations (prs) conferring a defect in protein secretion was cloned from a lambdaGEM-11 expression library. The sites of three closely linked prs mutations (prs-3, prs-29 and prs-40) were found to reside in a 5.3 kb DNA fragment, which also complemented the secretion defect in prs-3 and prs-29 mutants. Partial sequencing of the fragment showed that these three mutations affect one distinct gene (prsA) encoding a putative protein of 292 amino acids (33 kDa). Sequence analysis indicated the PrsA protein to be a lipoprotein located outside the cytoplasmic membrane. Thirty percent identity was shown to the PrtM protein of Lactococcus lactis, which is involved in the maturation of an exported proteinase. The phenotypes of prsA mutants and the structural similarity of PrsA with PrtM suggest that PrsA may have a novel function at a late phase in protein export.     

Human GLTP and mutant forms of ACD11 suppress cell death in the Arabidopsis acd11 mutant

The Arabidopsis acd11 mutant exhibits runaway, programmed cell death due to the loss of a putative sphingosine transfer protein (ACD11) with homology to mammalian GLTP. We demonstrate that transgenic expression in Arabidopsis thaliana of human GLTP partially suppressed the phenotype of the acd11 null mutant, resulting in delayed programmed cell death development and plant survival. Surprisingly, a GLTP mutant form impaired in glycolipid transfer activity also complemented the acd11 mutants. To understand the relationship between functional complementarity and transfer activity, we generated site-specific mutants in ACD11 based on homologous GLTP residues required for glycolipid transfer. We show that these ACD11 mutant forms are impaired in their in vitro transfer activity of sphingolipids. However, transgenic expression of these mutant forms fully complemented acd11 mutant cell death, and transgenic plants showed normal induction of hypersensitive cell death upon infection with avirulent strains of Pseudomonas syringae. The significance of these findings with respect to the function(s) of ACD11 in sphingolipid transport and cell death regulation is discussed.     

Investigation of the contribution of histidine 119 to the conduction of protons through human Nox2.

The conduction of protons through human Nox2 has previously been shown to be dependent upon His115. Alignment of sequences for both animal and plant Nox proteins indicated that histidines 115 and 119 are both highly conserved, while His111 was conserved among animal homologues of Nox1-4. To investigate the possible role that these histidine residues might play in the conduction of protons through Nox2, we have introduced both paired and single mutations into these histidine residues. Each construct was used to generate a CHO cell line in which the expression of the mutated Nox2 was assessed. Nox2 was expressed in each of the CHO cell lines generated, however, the level of expression of H111/115L in CHO cells was lower and that of H111L very much reduced, compared to that of wild-type Nox2. The arachidonic acid activated proton flux was absent in the CHO cell lines expressing the mutations of H111/115L, H111/119L or H115/119L, compared to that observed for wild-type Nox2. Similarly only a small efflux of protons was observed from CHO cells expressing either H119L or H111L. In all cases the expected proton flux was elicited through the addition of the protonophore, carbonyl cyanide m-chlorophenylhydrazone. Conclusions regarding the role of His111 in the conduction of protons cannot be drawn due to the reduced expression. We can, however, conclude that His119, in addition to His115, is required for the conduction of protons through Nox2. His119 has been identified as a highly conserved residue for which no function has previously been proposed.     

Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation

The effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures < 300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (deltaV++) were about -75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (deltaV++ approximately -60 ml/mol). The intrinsic fluorescence of TLN changed at pressures > 300 MPa, and the latter two mutants showed a smaller deltaGapp and deltaVapp of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region.     

褐飞虱表皮蛋白基因NlICP的克隆及功能研究

表皮蛋白与几丁质结合构成抵御外界不良环境的昆虫角质层, 在昆虫的生长发育及蜕皮硬化中具有重要的作用。为了探讨表皮蛋白基因在褐飞虱Nilaparvata lugens生长发育中的功能, 本研究根据褐飞虱转录组测序信息, 对其中1个预测为编码表皮蛋白的Unigene36450序列进行了克隆, 并应用荧光定量PCR和RNA干扰（RNAi）技术分别对该基因的表达规律和功能进行了研究。结果表明： 克隆的 Unigene36450全长cDNA开放阅读框长585 bp, 编码的蛋白含194个氨基酸, 具有典型的表皮蛋白R&R保守结构域, 命名为NlICP。转录水平的时序表达分析发现, NlICP仅在褐飞虱若虫期表达, 且在3龄若虫体内表达量最高, 提示该基因编码的蛋白属于幼虫表皮蛋白。RNA干扰结果显示, 取食dsNlICP的1龄末2龄初（孵化后第3 天）若虫在干扰6 d和8 d时, NlICP基因的表达量分别较取食dsGFP的对照组下降58.8%和45.6%, 差异极显著(P<0.01); 干扰后部分褐飞虱若虫因蜕皮不完全死亡, 干扰5 d的存活率较对照下降26.7%。本研究结果提示, NlICP与褐飞虱若虫的生长发育及蜕皮相关, 可以作为褐飞虱防治的潜在靶标基因。     

Different mutations in mucA gene of Pseudomonas aeruginosa mucoid strains in cystic fibrosis patients and their effect on algU gene expression

Alginate biosynthesis in Pseudomonas aeruginosa is a highly regulated process in which algU and mucA genes are key elements. Mutations in mucA gene determine alginate operon overexpression and exopolysaccharide overproduction. In our study, 119 strains of P. aeruginosa were isolated from sputa of 96 cystic fibrosis patients and 84/119 showed nonmucoid phenotype, while 35/119 showed mucoid phenotypes. mucA gene was amplified and sequenced in all strains revealing mutations in 29/35 mucoid strains (82%) and in one non-mucoid strain. 4/29 strains showed mutations never described that generated premature stop and much shorter MucA proteins. In all mutated strains, algU gene expression was analyzed to determine if mutations in mucA, resulting in a strong loss of its protein, could significantly influence its function and subsequently the biosynthetic pathways under algU control. Analysis of algU expression disclosed that the length significantly affects the expression of genes involved in the production of alginate and in the motility and hence survival of P. aeruginosa strains in cystic fibrosis lungs.     

Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli

Escherichia coli NZN111 is blocked in the ability to grow fermentatively on glucose but gave rise spontaneously to a mutant that had this ability. The mutant carries out a balanced fermentation of glucose to give approximately 1 mol of succinate, 0. 5 mol of acetate, and 0.5 mol of ethanol per mol of glucose. The causative mutation was mapped to the ptsG gene, which encodes the membrane-bound, glucose-specific permease of the phosphotransferase system, protein EIICB(glc). Replacement of the chromosomal ptsG gene with an insertionally inactivated form also restored growth on glucose and resulted in the same distribution of fermentation products. The physiological characteristics of the spontaneous and null mutants were consistent with loss of function of the ptsG gene product; the mutants possessed greatly reduced glucose phosphotransferase activity and lacked normal glucose repression. Introduction of the null mutant into strains not blocked in the ability to ferment glucose also increased succinate production in those strains. This phenomenon was widespread, occurring in different lineages of E. coli, including E. coli B.     

Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.     

Docking of the periplasmic FecB binding protein to the FecCD transmembrane proteins in the ferric citrate transport system of Escherichia coli

Citrate-mediated iron transport across the cytoplasmic membrane is catalyzed by an ABC transporter that consists of the periplasmic binding protein FecB, the transmembrane proteins FecC and FecD, and the ATPase FecE. Salt bridges between glutamate residues of the binding protein and arginine residues of the transmembrane proteins are predicted to mediate the positioning of the substrate-loaded binding protein on the transmembrane protein, based on the crystal structures of the ABC transporter for vitamin B(12), consisting of the BtuF binding protein and the BtuCD transmembrane proteins (E. L. Borths et al., Proc. Natl. Acad. Sci. USA 99:16642-16647, 2002). Here, we examined the role of the residues predicted to be involved in salt-bridge formation between FecB and FecCD by substituting these residues with alanine, cysteine, arginine, and glutamate and by analyzing the citrate-mediated iron transport of the mutants. Replacement of E93 in FecB with alanine [FecB(E93A)], cysteine, or arginine nearly abolished citrate-mediated iron transport. Mutation FecB(E222R) nearly eliminated transport, and FecB(E222A) and FecB(E222C) strongly reduced transport. FecD(R54C) and FecD(R51E) abolished transport, whereas other R-to-C mutations in putative interaction sites between FecCD and FecB substantially reduced transport. The introduced cysteine residues in FecB and FecCD also served to examine the formation of disulfide bridges in place of salt bridges between the binding protein and the transmembrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggest cross-linking of FecB(E93C) to FecD(R54C) and FecB(E222C) to FecC(R60C). The data are consistent with the proposal that FecB(E93) is contained in the region that binds to FecD and FecB(E222) in the region that binds to FecC.     

RNAi for plant functional genomics

A major challenge in the post-genome era of plant biology is to determine the functions of all the genes in the plant genome. A straightforward approach to this problem is to reduce or knock out expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study, but it is limited by gene redundancy, lethal knock-outs, nontagged mutants and the inability to target the inserted element to a specific gene. RNA interference (RNAi) of plant genes, using constructs encoding self-complementary 'hairpin' RNA, largely overcomes these problems. RNAi has been used very effectively in Caenorhabditis elegans functional genomics, and resources are currently being developed for the application of RNAi to high-throughput plant functional genomics.     

Mutational analysis of a role for salicylic acid in iron metabolism of Mycobacterium smegmatis

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc(2)155 and NCIMB 8548) and three mutant strains (mc(2)1292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of Mycobacterium smegmatis. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 microgram/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc(2)155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with p-aminosalicylate (PAS) at 100 microgram/ml increased salicylate accumulation between three- and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal.     

Active site mutants of human cyclophilin A separate peptidyl-prolyl isomerase activity from cyclosporin A binding and calcineurin inhibition.

Based on recent X-ray structural information, six site-directed mutants of human cyclophilin A (hCyPA) involving residues in the putative active site--H54, R55, F60, Q111, F113, and H126--have been constructed, overexpressed, and purified from Escherichia coli to homogeneity. The proteins W121A (Liu, J., Chen, C.-M., & Walsh, C.T., 1991a, Biochemistry 30, 2306-2310), H54Q, R55A, F60A, Q111A, F113A, and H126Q were assayed for cis-trans peptidyl-prolyl isomerase (PPIase) activity, their ability to bind the immunosuppressive drug cyclosporin A (CsA), and protein phosphatase 2B (calcineurin) inhibition in the presence of CsA. Results indicate that H54Q, Q111A, F113A, and W121A retain 3-15% of the catalytic efficiency (kcat/Km) of wild-type recombinant hCyPA. The remaining three mutants (R55A, F60A, and H126Q) each retain less than 1% of the wild-type catalytic efficiency, indicating participation by these residues in PPIase catalysis. Each of the mutants bound to a CsA affinity matrix. The mutants R55A, F60A, F113A, and H126Q inhibited calcineurin in the presence of CsA, whereas W121A did not. Although CsA is a competitive inhibitor of PPIase activity, it can complex with enzymatically inactive cyclophilins and inhibit the phosphatase activity of calcineurin.