Ruey-Fen Liou

Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site.

In Caenorhabditis elegans, pre‐mRNAs that are trans‐spliced are distinguished by the presence of an ‘outron’, intron‐like RNA at the 5′ end followed by a splice acceptor. We report that trans‐splicing of the rol‐6 gene can be completely suppressed simply by introducing a donor site into its 173 nt outron, at a site 50 nt upstream of the trans‐splice site, thereby converting rol‐6 into a conventional gene with a spliced intron near its 5′ end. When the consensus donor site was inserted at sites further upstream it was less effective in replacing transplicing with cis‐splicing. Surprisingly, the length of the intron was not the important variable, since lengthening of the 50 nt intron to 250 nt did not restore trans‐splicing. Apparently the context into which the splice site was introduced determined the efficiency of its use. These results support the conclusion that the sole signal for trans‐splicing is the presence of an outron. Clearly, cis‐ and trans‐splice acceptor sites are interchangeable, allowing the possibility of competition between the two types of splicing.

Functional Characterization of a Gene Family Encoding Polygalacturonases in Phytophthora parasitica

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of plant species. In our previous study, we discovered a multigene family encoding endopolygalacturonases (endoPG) in Phytophthora parasitica. Here, we screened the genomic library of Phytophthora parasitica for the genes encoding endoPG named pppg2 through pppg10, and analyzed their functions. Results obtained by real-time quantitative reverse transcriptase-polymerase chain reaction demonstrated that some of these genes are highly induced during plant infection, which suggests their important roles in the pathogenesis of Phytophthora parasitica. Analysis by in-gel activity assay of recombinant proteins obtained from Pichia pastoris indicated that each of these genes encodes a functional endoPG. Investigation of the function of pppg genes in planta by a Potato virus X agroinfection system in tobacco revealed that each pppg caused specific effects, varying from no symptoms to dwarfism, necrosis, leaf curl, silvery leaf, and cracks in leaf stalks. Appearance of these effects depends on the expression of a pppg protein with a normal active site in the apoplast. These results indicated that each pppg plays a distinct role in the decomposition of plant cell wall.

The period gene of the German cockroach and its novel linking power between vertebrate and invertebrate.

Circadian clock protein PERIOD (PER) is essential for the endogenous clockworks in diverse lineages within Metazoa, but the protein sequences, the clock protein interactions, and the photic responses are variant and different between vertebrate and invertebrate PER homologs. Here we identified the German cockroach PER homologs and found it could bridge the huge phylogenetic gap and make possible a more precise protein sequence comparison between vertebrate and invertebrate PER homologs. Seven blocks of conserved regions (c1–c7) interspersed within PER proteins were defined, and two new significant homologies were found in the upstream portion of c3 region and in the c7 region, respectively. In addition, we found all dipteran insects PER homologs lack the c7 region and its following amino acid residues. Our results not only reveal the homology and divergence, but also imply the constraint and plasticity of divergent PER proteins during the course of evolution. These findings lay a solid foundation for understanding the general and divergent properties of circadian clockworks in diverse lineages within Metazoa.

A novel elicitor protein from Phytophthora parasitica induces plant basal immunity and systemic acquired resistance

The interaction between Phytophthora pathogens and host plants involves the exchange of complex molecular signals from both sides. Recent studies of Phytophthora have led to the identification of various apoplastic elicitors known to trigger plant immunity. Here, we provide evidence that the protein encoded by OPEL of Phytophthora parasitica is a novel elicitor. Homologues of OPEL were identified only in oomycetes, but not in fungi and other organisms. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that OPEL is expressed throughout the development of P. parasitica and is especially highly induced after plant infection. Infiltration of OPEL recombinant protein from Escherichia coli into leaves of Nicotiana tabacum (cv. Samsun NN) resulted in cell death, callose deposition, the production of reactive oxygen species and induced expression of pathogen-associated molecular pattern (PAMP)-triggered immunity markers and salicylic acid-responsive defence genes. Moreover, the infiltration conferred systemic resistance against a broad spectrum of pathogens, including Tobacco mosaic virus, the bacteria wilt pathogen Ralstonia solanacearum and P. parasitica. In addition to the signal peptide, OPEL contains three conserved domains: a thaumatin-like domain, a glycine-rich protein domain and a glycosyl hydrolase (GH) domain. Intriguingly, mutation of a putative laminarinase active site motif in the predicted GH domain abolished its elicitor activity, which suggests enzymatic activity of OPEL in triggering the defence response.

Interaction of snake venom cardiotoxin (a membrane-disruptive polypeptide) with human erythrocytes

The action of 7.2 µM cardiotoxin on 0.25% human erythrocytes in a plasma extender solution was studied by the interaction of toxin with intact red blood cells and subsequent hemolysis of the cells. The binding of toxin to cells was completed within 10 min, whereas the membrane rigidity was weakened in a non-lytic period for about 25 min. The toxin molecules bound almost exclusively to the membrane. The bound toxin could not be liberated with either 0.5% Triton X-100 or 0.1 N NaOH. The degree of binding was slightly reduced in the presence of 10 mM mono- and divalent inorganic salts. The action of toxin might weaken the in situ association of several proteins that are linked with band 3 protein of the membrane, thus making the cells fragile and altering the shape of the cell to a smooth sphere.

Tomato SOBIR1/EVR Homologs Are Involved in Elicitin Perception and Plant Defense Against the Oomycete Pathogen Phytophthora parasitica.

During host-pathogen interactions, pattern recognition receptors form complexes with proteins, such as receptor-like kinases, to elicit pathogen-associated molecular pattern-triggered immunity (PTI), an evolutionarily conserved plant defense program. However, little is known about the components of the receptor complex, as are the molecular events leading to PTI induced by the oomycete Phytophthora pathogen. Here, we demonstrate that tomato (Solanum lycopersicum) SlSOBIR1 and SlSOBIR1-like genes are involved in defense responses to Phytophthora parasitica. Silencing of SlSOBIR1 and SlSOBIR1-like enhanced susceptibility to P. parasitica in tomato. Callose deposition, reactive oxygen species production, and PTI marker gene expression were compromised in SlSOBIR1- and SlSOBIR1-like-silenced plants. Interestingly, P. parasitica infection and elicitin (ParA1) treatment induced the relocalization of SlSOBIR1 from the plasma membrane to endosomal compartments and silencing of NbSOBIR1 compromised ParA1-mediated cell death on Nicotiana benthamiana. Moreover, the SlSOBIR1 kinase domain is indispensable for ParA1 to trigger SlSOBIR1 internalization and plant cell death. Taken together, these results support the idea of participation of solanaceous SOBIR1/EVR homologs in the perception of elicitins and indicate their important roles in plant basal defense against oomycete pathogens.

Complete nucleotide sequence of Beauveria bassiana 5.8s rRNA coding gene and flanking internal transcribed spacers

The nucleotide sequence of two clones of Beauveria bassiana in 5.8s rRNA coding gene and ITS regions were completely sequenced. The overall sequence similarity of these two clones is 96%. The identities of internal transcribed spacer (ITS) regions are 91 % (ITSI) and 100% (ITSII), respectively. Both of 5.8s rRNA sequences have 98% homology.

Complete nucleotide sequence and genome organization of a Cactus virus X strain from Hylocereus undatus (Cactaceae).

Summary.The complete nucleotide sequence of a strain of Cactus virus X (CVX-Hu) isolated from Hylocereus undatus (Cactaceae) has been determined. Excluding the poly(A) tail, the sequence is 6614 nucleotides in length and contains seven open reading frames (ORFs). The genome organization of CVX is similar to that of other potexviruses. ORF1 encodes the putative viral replicase with conserved methyltransferase, helicase, and polymerase motifs. Within ORF1, two other ORFs were located separately in the +2 reading frame, we call these ORF6 and ORF7. ORF2, 3, and 4, which form the “triple gene block” characteristic of the potexviruses, encode proteins with molecular mass of 25, 12, and 7 KDa, respectively. ORF5 encodes the coat protein with an estimated molecular mass of 24 KDa. Sequence analysis indicated that proteins encoded by ORF1-5 display certain degree of homology to the corresponding proteins of other potexviruses. Putative product of ORF6, however, shows no significant similarity to those of other potexviruses. Phylogenetic analyses based on the replicase (the methyltransferase, helicase, and polymerase domains) and coat protein demonstrated a closer relationship of CVX with Bamboo mosaic virus, Cassava common mosaic virus, Foxtail mosaic virus, Papaya mosaic virus, and Plantago asiatica mosaic virus.

Generation and Analysis of the Expressed Sequence Tags from the Mycelium of Ganoderma lucidum

Ganoderma lucidum (G. lucidum) is a medicinal mushroom renowned in East Asia for its potential biological effects. To enable a systematic exploration of the genes associated with the various phenotypes of the fungus, the genome consortium of G. lucidum has carried out an expressed sequence tag (EST) sequencing project. Using a Sanger sequencing based approach, 47,285 ESTs were obtained from in vitro cultures of G. lucidum mycelium of various durations. These ESTs were further clustered and merged into 7,774 non-redundant expressed loci. The features of these expressed contigs were explored in terms of over-representation, alternative splicing, and natural antisense transcripts. Our results provide an invaluable information resource for exploring the G. lucidum transcriptome and its regulation. Many cases of the genes over-represented in fast-growing dikaryotic mycelium are closely related to growth, such as cell wall and bioactive compound synthesis. In addition, the EST-genome alignments containing putative cassette exons and retained introns were manually curated and then used to make inferences about the predominating splice-site recognition mechanism of G. lucidum. Moreover, a number of putative antisense transcripts have been pinpointed, from which we noticed that two cases are likely to reveal hitherto undiscovered biological pathways. To allow users to access the data and the initial analysis of the results of this project, a dedicated web site has been created at http://csb2.ym.edu.tw/est/.

Molecular evidence that aphid-transmitted Alpinia mosaic virus is a tentative member of the genus Macluravirus

Summary. Alpinia mosaic virus (AlpMV), once assigned to the genus Potyvirus, infects primarily plants of the ginger family. To seek molecular evidence for correct classification of this virus, a cDNA clone corresponding to the 3′ portion of the AlpMV genome was obtained by reverse transcriptase-PCR and TA cloning. The authenticity of the cDNA clone was confirmed by expression of the coat protein (CP) in E. coli followed by immunoblot analysis. Sequence analysis indicated that, in contrast to its low identity with all the other genera of the family Potyviridae, the deduced amino acid sequence of AlpMV CP was 42.9 ∼ 61.9% identical to members of the genus Macluravirus. Phylogenetic analysis also demonstrated that the AlpMV CP clustered with those of Cardamom mosaic virus and Chinese yam necrotic mosaic virus. These results indicate that AlpMV should be classified as a tentative species within the genus Macluravirus rather than Potyvirus as proposed previously.