Induction of thaumatin‐like proteins (TLPs) in Rhizoctonia solani‐infected rice and characterization of two new cDNA clones

Thaumatin‐like proteins (TLPs) were shown to be induced in rice plants (cv. IR58) that were infected with the sheath blight fungus, Rhizoctonia solani . Western blot analysis revealed the presence of two TLPs with sizes of 25 and 24 kDa which are different from a previously reported TLP with a size of 15.6 kDa from rice plants infiltrated with the non‐pathogenic bacterium, Pseudomonas syringae pv. syringae . By probing a cDNA expression library prepared from RNA isolated from R. solani ‐infected rice plants with a TLP antibody, several putative TLP cDNA clones were isolated and sequenced. The cDNA clones appeared to be derived from two different genes which shared only 77% sequence identity with each other and a lower percentage of sequence identity with the previously reported TLP cDNA clone. Southern blot analysis with the two TLP cDNAs revealed different rice genomic DNA fragments. Northern blot analysis also confirmed that a 1.1‐kb RNA detectable by the TLP cDNA inserts was induced by fungal infection. Thus rice TLPs are encoded by a family of at least three genes which are differentially expressed in responses to bacterial or fungal pathogens.     

水稻类Tubby蛋白质在叶片生长和白叶枯病抗性反应中的表达

类Tubby蛋白质（Tubby-like protein,TLP）在动植物中广泛存在,暗示其在生命过程中发挥重要的作用。水稻（Oryza sativa）基因组中有14个TLP家族成员,首先制备了这些蛋白质的抗体,用免疫印迹方法检测了它们在水稻叶片不同生长时期的表达情况,揭示其表达模式;然后对Xa21介导的水稻白叶枯病抗性反应不同时间点进行检测,发现OsTLP2、OsTLP7、OsTLP8和OsTLP9等4个蛋白质的表达发生了变化;进一步比较它们在抗病、感病反应和对照处理中的表达情况,发现不同反应间的表达也有区别。该研究结果为阐释水稻TLP在叶片生长过程中的功能,尤其是在水稻-白叶枯病菌互作过程中的作用提供了重要线索。     

The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function

Thaumatin-like proteins (TLPs) are the products of a large, highly complex gene family involved in host defence and a wide range of developmental processes in fungi, plants, and animals. Despite their dramatic diversification in organisms, TLPs appear to have originated in early eukaryotes and share a well-defined TLP domain. Nonetheless, determination of the roles of individual members of the TLP superfamily remains largely undone. This review summarizes recent advances made in elucidating the varied TLP activities related to host resistance to pathogens and other physiological processes. Also discussed is the current state of knowledge on the origins and types of TLPs, regulation of gene expression, and potential biotechnological applications for TLPs.     

Structural and functional evolution of transthyretin and transthyretin-like proteins

Transthyretin (TTR) is a tetrameric protein involved in the distribution of thyroid hormones in vertebrates. The amino acid sequence of TTR is highly conserved across vertebrates. Hypothetical TTR-like proteins (TLPs) were inferred from the identification of genes in nonvertebrate species. Here, we identified five motifs defining TLPs and three motifs defining both TTRs and TLPs. These motifs were mapped onto structurally conserved and functionally important regions of TTRs. These motifs were used to build hidden Markov models for accurate identification of TLPs in other organisms. TLPs were divided into three main groups based on their N-terminal regions. Most TLPs are cytosolic, but in plants and slime mold, we predict they are peroxisomal. We verified that the TLPs from enterobacteria were periplasmic. We demonstrated that TLP genes are expressed in a bacterium (E. coli), an invertebrate animal (C. elegans), and a plant (A. thaliana). These TLPs have similar subunit molecular weights to TTRs, are tetramers, and are predicted to have similar three-dimensional (3D) structures to TTRs, but do not bind thyroid hormones or similar ligands. We suggest that like TTRs, the N-terminal and C-terminal regions of TLPs are integral in defining the function of TLPs in nonvertebrate species and that the TLP gene duplicated in primitive vertebrates to produce the TTR gene. TLP/TTR has retained its overall structure, but changed function and localization during evolution in bacteria, invertebrates, plants, and vertebrates.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

Genome-wide identification,characterization and expression analysis of the TLP gene family in melon (Cucumis melo L.)

Thaumatin-like proteins (TLPs), which belong to pathogenesis-related (PR) protein family 5 (PR5), are involved in plant host defense and various developmental processes. The functions of the TLP family have been extensively discussed in multiple organisms, whereas the detailed information of this family in melon has not been reported yet. In this study, we identified 28 TLP genes in the melon genome and a N-terminal signal peptide was found highly conserved within each member of this family. Phylogeny analysis indicated that TLPs from melon and other plant species were clustered into ten groups. Twelve segmental and seven tandem duplication gene pairs that underwent purifying selection were identified. TLP genes expressed differentially in different tissues/organs, and were significantly induced after Podosphaera xanthii infection. TLPs in breeding line MR-1 tend to express early after pathogen infection compared with cultivar Top Mark. Our study provides a comprehensive understanding of the melon TLP family and demonstrates their potential roles in disease resistance, therefore provides more reference for further research.     

Structural and functional characteristics of plant proteinase inhibitor-II (PI-II) family

Plant proteinase inhibitor-II (PI-II) proteins are one of the promising defensive proteins that helped the plants to resist against different kinds of unfavorable conditions. Different roles for PI-II have been suggested such as regulation of endogenous proteases, modulation of plant growth and developmental processes and mediating stress responses. The basic knowledge on genetic and molecular diversity of these proteins has provided significant insight into their gene structure and evolutionary relationships in various members of this family. Phylogenetic comparisons of these family genes in different plants suggested that the high rate of retention of gene duplication and inhibitory domain multiplication may have resulted in the expansion and functional diversification of these proteins. Currently, a large number of transgenic plants expressing PI-II genes are being developed for enhancing the defensive capabilities against insects, bacteria and pathogenic fungi. Much emphasis is yet to be given to exploit this ever expanding repertoire of genes for improving abiotic stress resistance in transgenic crops. This review presents an overview about the current knowledge on PI-II family genes, their multifunctional role in plant defense and physiology with their potential applications in biotechnology.     

Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.)

Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.     

Analyses of phylogeny, evolution, conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors

Background and Aims

The cell cycle is controlled by cyclin-dependent kinases (CDKs), and CDK inhibitors are major regulators of their activities. The ICK/KRP family of CDK inhibitors has been reported in several plants, with seven members in arabidopsis; however, the phylogenetic relationship among members in different species is unknown. Also, there is a need to understand how these genes and proteins are regulated. Furthermore, little information is available on the functional differences among ICK/KRP family members.

Methods

We searched publicly available databases and identified over 120 unique ICK/KRP protein sequences from more than 60 plant species. Phylogenetic analysis was performed using 101 full-length sequences from 40 species and intron–exon organization of ICK/KRP genes in model species. Conserved sequences and motifs were analysed using ICK/KRP protein sequences from arabidopsis (Arabidopsis thaliana), rice (Orysa sativa) and poplar (Populus trichocarpa). In addition, gene expression was examined using microarray data from arabidopsis, rice and poplar, and further analysed by RT-PCR for arabidopsis.

Key Results and Conclusions

Phylogenetic analysis showed that plant ICK/KRP proteins can be grouped into three major classes. Whereas the C-class contains sequences from dicotyledons, monocotyledons and gymnosperms, the A- and B-classes contain only sequences from dicotyledons or monocotyledons, respectively, suggesting that the A- and B-classes might have evolved from the C-class. This classification is also supported by exon–intron organization. Genes in the A- and B- classes have four exons, whereas genes in the C-class have only three exons. Analysis of sequences from arabidopsis, rice and poplar identified conserved sequence motifs, some of which had not been described previously, and putative functional sites. The presence of conserved motifs in different family members is consistent with the classification. In addition, gene expression analysis showed preferential expression of ICK/KRP genes in certain tissues. A model has been proposed for the evolution of this gene family in plants.     

Steroid receptor co-activator is required for juvenile hormone signal transduction through a bHLH-PAS transcription factor, methoprene tolerant

Metamorphosis in insects is regulated by juvenile hormone (JH) and ecdysteroids. The mechanism of 20-hydroxyecdysone (20E), but not of JH action, is well understood. A basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family member, methoprene tolerant (Met), plays an important role in JH action. Microarray analysis and RNA interference (RNAi) were used to identify 69 genes that require Met for their hydroprene-regulated expression in the red flour beetle, Tribolium castaneum. Quantitative real time PCR analysis confirmed microarray data for 13 of the 16 hydroprene-response genes tested. The members of the bHLH-PAS family often function as heterodimers to regulate gene expression and Met is a member of this family. To determine whether other members of the bHLH-PAS family are required for the expression of JH-response genes, we employed RNAi to knockdown the expression of all 11 members of the bHLH-PAS family and studied the expression of JH-response genes in RNAi insects. These studies showed that besides Met, another member of this family, steroid receptor co-activator (SRC) is required for the expression of 15 JH-response genes tested. Moreover, studies in JH responsive Aag-2 cells revealed that Aedes aegypti homologues of both Met and SRC are required for the expression of the JH-response gene, kr-h1, and SRC is required for expression of ecdysone-response genes. These data suggest the steroid receptor co-activator plays key roles in both JH and 20E action suggesting that this may be an important molecule that mediates cross-talk between JH and 20E to prevent metamorphosis.     

Molecular evolutionary analyses of the Arabidopsis L7 ribosomal protein gene family

Cytoplasmic ribosomal protein (r-protein) genes in Arabidopsis thaliana are encoded by 80 multigene families that contain between two and seven members. Gene family members are typically similar at the protein sequence level, with the most divergent members of any gene family retaining 94% identity, on average. However, three Arabidopsis r-protein families - S15a, L7 and P2 - contain highly divergent family members. Here, we investigated the organization, structure, expression and molecular evolution of the L7 r-protein family. Phylogenetic analyses showed that L7 r-protein gene family members constitute two distinct phylogenetic groups. The first group including RPL7B, RPL7C and RPL7D has homologs in plants, animals and fungi. The second group represented by RPL7A is found in plants but has no orthologs from other fully-sequenced eukaryotic genomes. These two groups may have derived from a duplication event prior to the divergence of animals and plants. All four L7 r-protein genes are expressed and all exhibit a differential expression in inflorescence and flowers. RPL7A and RPL7B are less expressed than the other genes in all tissues analyzed. Molecular characterization of nucleic and protein sequences of L7 r-protein genes and analysis of their codon usage did not indicate any functional divergence. The probable evolution of an extra-ribosomal function of group 2 genes is discussed.     

Evolution of Cinnamate/p-coumarate carboxyl methyltransferases and their role in the biosynthesis of methylcinnamate

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.     

Genome-scale identification of MLO domain-containing genes in soybean (Glycine max L. Merr.)

In plants, powdery-mildew-resistance locus o (Mlo) genes encode proteins that are calmodulin-binding proteins involved in a variety of cellular processes. However, systematic characterization of this gene family in soybean (Glycine max L. Merr.) has not been yet reported. In this study, we identified MLO domain-contained members in soybean and examined their expression under phytohormone treatment and abiotic stress conditions. A total of 20 soybean Mlo genes were identified (GmMlo1-20), which are distributed on 13 chromosomes, and display diverse exon-intron structures. Phylogenetic analysis indicated that the Mlo family can be classified into four subfamilies. Sequence comparison was used to reveal the conserved calmodulin-binding domain (CaMBD) in GmMLO proteins. The expression of GmMlo genes was influenced by various phytohormone treatments and abiotic stresses, suggesting that these Mlo genes have various roles in the response of soybean to environmental stimuli. Promoter sequence analysis revealed an overabundance of stress and/or phytohormone-related cis-elements in GmMlo genes. These data provide important clues for elucidating the functions of genes of the Mlo gene family.     

Expression of a subset of the Arabidopsis Cys(2)/His(2)-type zinc-finger protein gene family under water stress

The genes encoding Cys(2)/His(2)-type zinc-finger proteins constitute a large family in higher plants. To elucidate the functional roles of these types of protein, four different members of the gene family were cloned from Arabidopsis by PCR-aided methods. One was identical to the already reported gene STZ/ZAT10 and three were as yet unidentified genes, then designated AZF1 (Arabidopsis zinc-finger protein 1), AZF2 and AZF3. The AZF- and STZ-encoded proteins contain two canonical Cys(2)/His(2)-type zinc-finger motifs, separated by a long spacer. Three conserved regions, named B-box, L-box, and DNL-box, were also recognized outside the zinc-finger motifs, as in other members of the two-fingered Cys(2)/His(2)-type zinc-finger protein family. These four genes were positioned on the same branch of a phylogenetic tree constructed based on the zinc-finger motif sequences, suggesting their structural and functional relationship. RNA blot analysis showed that all four genes were mainly expressed in roots and at different levels in other organs. Expression of the four genes responded to water stress. High-salt treatment resulted in elevated levels of expression of all of these genes. Low-temperature treatment increased the expression levels of AZF1, AZF3, and STZ, but not AZF2. Only AZF2 expression was strongly induced by ABA treatment, where the time course of the induction was similar to that caused by high salinity. In situ localization showed that AZF2 mRNA accumulated in the elongation zone of the roots under the salt-stress condition. These results suggest that AZF1, AZF2, AZF3, and STZ are all involved in the water-stress response in an ABA-dependent or -independent pathway to regulate downstream genes.