植物几丁质酶与病害防治研究进展

植物与病原物相互作用的关系问题,是植物病理学的中心内容之一,也是近年来进展最快的领域,其中有关植物病程相关蛋白(PR蛋白)的研究尤为引人注目。 为了对付病原物的入侵,植物通过进化形成了一整套复杂的、在不同水平上起作用的防卫系统。植物的一种重要的防卫系统是过敏性反应(HR反应);过敏性反应的结果形成植物的诱导抗性,包括局限于侵染点的局部诱导抗性和除侵染点外整株植物的系统性诱导抗性。诱导抗性在多种植物中已有报道,其生理生化基础正在逐步被阐明。     

PR—蛋白与高等植物的诱导抗性

一某些过敏性寄主植物在感染病毒或其它病原物后,以牺牲局部叶片(在接种叶片上形成局部枯斑或坏死)而使全侏免遭罹难,当相关性或非相关性病原物再次侵染时,植株即表面出抗性。这种抗性不同于植物本身所固有的先天抗性,由于它是在受病原物侵染(或化学药物处理)后诱导产生的,故称之为诱导抗性(或获得抗性)。诱导抗性的共同特征是作用的非持异性,即由病毒诱导的抗性不仅可以抵抗非相关病毒的侵染,还可以抵抗     

丛枝菌根菌诱导植物抗病的内在机制

应用菌根真菌诱导植物抗病性是近年化学生态学和病害生物防治研究的热点．研究表明,丛枝菌根真菌(AMF)对土传病原物具有一定拮抗或抑制作用,能提高植物对土传病害的抗／耐病性．在菌根根际,各种菌群不断产生相互作用,AMF在其中起着抑制病原菌、促进有益菌生长的作用,可与其他桔抗菌结合,用做生防菌．AMF提高植物抗病性的机制还有这样几种假设：(1)植物营养得到改善;(2)竞争作用;(3)根系形态结构改变;(4)根际微生物区系变化;(5)诱导抗性及诱导系统抗性,即AMF侵染植物根系后,诱导植物体内酚酸类代谢产物增加,使植物产生局部或系统防御反应．深人研究AMF提高植物抗病性的机制,有助于正确理解菌根的抗病作用,使其能尽快地成为植物病害生物防治中的一种新方法,在生态农业中发挥作用。     

生物源蛋白激发子的研究进展

生物源蛋白激发子是一类能诱导植物产生防卫反应的特殊化合物,主要来源于病原微生物、其他微生物及寄主植物或由寄主-病原物互作后产生。病原微生物或其他微生物产生的激发子包括真菌的β-葡聚糖、糖蛋白、脂类物质和其他细胞壁组分;由寄主植物产生的激发子主要是细胞壁组分中的寡糖物质,如寡聚半乳糖醛酸和木聚糖片段;寄主-病原物互作后产生的激发子主要是互作过程中酶对寄主和病原物细胞组分修饰后产生的。生物源蛋白激发子与寄主植物作用后,通过一系列信号传导,诱导寄主植物产生乙烯、植保素、水杨酸、茉莉酸、病程相关蛋白等,导致植物中多种防卫反应的发生,从而可以控制病害的发展和传播,在农业生产上能够起到减少病虫危害达到增产的目的。近年来,人们对激发子的研究非常广泛,生物源蛋白激发子在生物防治中的作用也日益受到学者们的重视。该文就生物源蛋白激发子的种类:Harpin蛋白、Nep1-like蛋白家族、RXLR蛋白家族、Elicitins及其各类型激发子的功能、信号传导和作用机制的研究进展情况和在农业中的应用进行了综述,并提出了生物源蛋白激发子将来在农业生产中对病害防治方面的展望。     

水杨酸诱导的烟草抗性相关序列的分离

通过已分离鉴定的水杨酸诱导烟草‘云烟85’中与抗性相关的差异表达基因,采用差示筛选和反式Northern检测以及序列分析得到94个烟草差异表达的EST序列。经测序及同源性比较,其中87个有同源序列,7个为新序列;有51个与抗性相关,占总序列的54.3%,其中有系统获得抗性蛋白基因和病程相关蛋白基因等。     

植物抗病反应的信号传导网络

植物由抗病基因介导的防卫过程存在一系列生理生化和分子生物学反应,这些反应从病原菌侵染点开始的超敏反应(HR)并延伸到远处组织的系统抗性或获得性抗性(SAR),受制于一种信号传导网络的调控。这个信号系统由抗病蛋白和病原菌非毒性蛋白在一种配体-受体的互作模式下激发,并由信号分子H2O2,NO和系统信号分子SA,JA和乙烯和通过关键调控基因传递和放大,最终诱导一系列防卫反应基因的表达和代谢的变化而产生抗性。植物防卫信号的产生有类似于动物免疫系统因子的介导,并可由非寄主病原菌或诱导子诱发。这些信号途径所产生的广谱抗性为植物抗病基因工程的应用奠定了基础。     

植物抗病反应的信号传导网络

植物由抗病基因介导的防卫过程存在一系列生理生化和分子生物学反应,这些反应从病原菌侵染点开始的超敏反应（HR）并延伸到远处组织的系统抗性或获得性抗性（SAR）,受制于一种信号传导网络的调控,这个信号系统由抗病蛋白和病原菌非毒性蛋白在一种配体-受体的互作模式下激发,并由信号分子H2O2,NO和系统信号分子SA,JA和乙烯和通过关键调控基因传递和放大,最终诱导一系列防卫反应基因的表达和代谢的变化而产生抗性。植物防卫信号的产生有类似于动物免疫系统因子的介导,并可由非寄主病原菌或诱导子诱发,这些信号途径所产生的广谱抗性为植物抗病基因工程的应用奠定了基础。     

植物系统获得性抗性的信号转导

坏死病原菌（necrotizingpathogen）的侵染或者一些化学因子的处理能诱导植物的非侵染或非处理部位产生对多种病原再侵染产生抗性,即系统获得性抗性（systemicacquiredresistance,SAR）。获得系统抗性的组织中SAR基因产物的累积和防卫反应的潜在诱导增强（potentiation）是其两类抗病机制。SAR至少有通过水杨酸（salicylicacid,SA）或茉莉酸（jasmonicacid,JA）、乙烯（ethylene）为系统信号分子的两类信号转导途径。遗传分析已用于SAR产生的信号转导过程的分析,一些与SAR信号转导相关的基因已经和正在克隆,这些基因具有明显提高植物广谱抗性的潜能。     

水稻类病斑突变体研究进展

植物类病变突变体是一类在没有病原物侵染情况下就能自发产生坏死斑的突变体。这类突变往往导致植株的抗病增强和防御相关基因的组成性表达。水稻中已报道了将近200个来源不同的类病变突变体,截至2014年12月73个水稻类病变突变体已被鉴定和命名,其中11个控制类病变性状的基因被克隆,它们分别编码不同的蛋白,包括热激蛋白转录因子、E3泛素连接酶、质膜蛋白激酶、锌指蛋白、酰基转移酶。尽管这些蛋白不是直接与植物抗病途径相关,但是在已鉴定的水稻类病变突变体中,绝大多数提高了对白叶枯病或稻瘟病的抗性,表明这些类病变基因的突变激活了植株的防御系统,并且不同的类病变基因可能参与了不同的抗病信号传导途径。深入研究水稻类病变突变体对作物抗病的分子机理研究和栽培品种的遗传改良都具有重要的意义。     

植物抗病分子机制研究进展

植物抗病机制是目前研究的热点。在长期的进化过程中,植物形成了一系列复杂有效的防御机制来抵御、破坏病原物的侵染。植物抗病基因在植物抗性反应中起着重要的作用,植物一旦监测到病原物马上起始防御反应,并伴随着植物体内一系列细胞和生理生化的变化。近年来,基因沉默作为一个重要的细胞内防御外源核酸的机制,越来越受到科学家重视。综述了植物抗病基因和基因沉默机制在植物抗病反应中的重要作用,并对研究植物抗病机制的前景做了展望。     

Proline-rich proteins and glycoproteins: expression of salivary gland multigene families

Our recent research interests have focused on a group of unusual proteins and glycoproteins high in proline content, or the so-called proline-rich proteins (PRPs). The PRPs are tissue-specific expressions of salivary gland multigene families. Normally PRPs are not detected or are present in very low amounts in rat, mouse and hamster salivary glands, but these unusual proteins are dramatically induced by treatment with the catecholamine isoproterenol. The structures and organizations of several PRP mRNAs and PRP genes have been determined. The amino acid sequences of all PRPs show 4 distinct regions, namely, a signal peptide, a transition region, a repeat region and a carboxyl-terminal region. Glycoproteins induced by isoproterenol treatment may be N-glycosylated or O-glycosylated. The N-glycosylated glycoprotein GP-158 from rat submandibular glands has a 12 amino acid glycopeptide which repeats possibly 49 times. Proline-rich proteins of the parotid glands of rats and mice are also greatly induced by dietary tannins. The apparent unique occurrence of PRPs in saliva suggests that one biological role is to neutralize the detrimental effects of dietary tannins and other polyphenols. The upstream regions of the mouse and hamster PRP genes contain cyclic AMP-regulated sequences as demonstrated by deletions and transient transfections. The PRP multigene family members of mouse are all located on chromosome 8.     

Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands

Summary Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with -amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.     

Structure, organization, and regulation of a hamster proline-rich protein gene. A multigene family

Genomic DNA fragments bearing proline-rich protein (PRP) genes expressed specifically in hamster parotid glands have been isolated and characterized. Complete exonic sequences as well as intronic and a considerable portion of the flanking sequences are reported for a PRP gene, H29. H29 is interrupted by three intervening sequences, with consensus splice junctions, and it likely encodes the acidic hamster PRP Hp43a. Exceedingly high homology of the 5'-untranslated region and the sequence encoding the signal peptide is observed with other PRPs of all species studied. Significant homology was also detected among the repetitive sequences of the mature acidic PRPs from human, mouse, hamster, and rat. This conservation of the internal repeats of the PRPs suggested that proline-rich protein gene evolution involved intragenic duplication of internal repeats and gene duplication and conversion. Both hamster and mouse PRP genes (H29 and mouse proline-rich protein gene, respectively) share considerable sequence similarity in the 5'-flanking regions for about 100 base pairs upstream. The remainder of the upstream sequences were heterologous except for three oligonucleotide regions with 60-70% sequence conservation. These three regions are thought to be involved in the regulation of the tissue-specific PRP gene induction.     

PRPs localized to the middle lamellae are required for cortical tissue integrity in Medicago truncatula roots

Key message

A family of repetitive proline-rich proteins interact with acidic pectins and play distinct roles in legume root cell walls affecting cortical and vascular structure.

Abstract

A proline-rich protein (PRP) family, composed of tandemly repeated Pro-Hyp-Val-X-Lys pentapeptide motifs, is found primarily in the Leguminosae. Four distinct size classes within this family are encoded by seven tightly linked genes: MtPRP1, MtPRP2 and MtPRP3, and four nearly identical MtPRP4 genes. Promoter fusions to β-glucuronidase showed strong expression in the stele of hairy roots for all 4 PRP genes tested, with additional expression in the cortex for PRP1, PRP2 and PRP4. All except MtPRP4 are strongly expressed in non-tumorous roots, and secreted and ionically bound to root cell walls. These PRPs are absent from root epidermal cell walls, and PRP accumulation is highly localized within the walls of root cortical and vascular tissues. Within xylem tissue, PRPs are deposited in secondary thickenings where it is spatially exclusive to lignin. In newly differentiating xylem, PRPs are deposited in the regularly spaced paired-pits and pit membranes that hydraulically connect neighboring xylem elements. Hairpin-RNA knock-down constructs reducing PRP expression in Medicago truncatula hairy root tumors disrupted cortical and vascular patterning. Immunoblots showed that the knockdown tumors had potentially compensating increases in the non-targeted PRPs, all of which cross-react with the anti-PRP antibodies. However, PRP3 knockdown differed from knockdown of PRP1 and PRP2 in that it greatly reduced viability of hairy root tumors. We hypothesize that repetitive PRPs interact with acidic pectins to form block-copolymer gels that can play distinct roles in legume root cell walls.

     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.     

Repurposing an Ancient Protein Core Structure: Structural Studies on FmtA,a Novel Esterase of Staphylococcus aureus

FmtA is a penicillin-recognizing protein (PRP) with novel hydrolytic activity toward the ester bond between d-Ala and the backbone of teichoic acids. Teichoic acids are polyol-phosphate polymers found in the Staphylococcus aureus cell wall, and they play important roles in antibiotic resistance and pathogenesis. Two of the PRPs conserved motifs, namely, SXXK and Y(S)XN, are involved in the hydrolysis by FmtA, but the catalytic mechanism remains elusive. Here we determined the crystal structure of FmtA. FmtA shares the core structure of PRPs: an all α-helical domain and α/β domain sandwiched together. However, it does not have the typical PRPs active-site cleft. Its active site is shallow, solvent-exposed, and enlarged. Furthermore, our mutagenesis and kinetic studies suggest that the SXXK and Y(S)XN motifs of FmtA offer all that is necessary for catalysis, and more: the active-site nucleophile (serine), the general base (lysine) required for the acylation step and the deacylation step, and an anchor (tyrosine) to hold the active-site serine, and possibly the substrate, in an optimum conformation for catalysis. Our study establishes that the FmtA esterase activity represents an expansion of the catalytic activity repertoire of the PRPs core structure, which typically displays peptidase activity. This finding points toward a novel mechanism of ester bond hydrolysis by a PRP. The structure of FmtA provides insights to the design of inhibitor molecules with the potential to serve as leads in the development of novel antibacterial chemotherapeutic agents.     

Localization of repetitive proline-rich proteins in the extracellular matrix of pea root nodules

Summary Early responses of legume roots toRhizobium inoculation include new cell wall synthesis and induction of some putative wall protein genes. Although the predicted amino acid sequences of several early nodulins indicate that they encode proline-rich proteins (PRPs), the proteins have been neither isolated nor has their presence been demonstrated in cell walls. We have used polyclonal antibodies against PRP2 from soybean to identify and localize proline-rich proteins in pea nodules. On immunoblots, several PRPs were detected, ranging from less than 20 kDa to 110 kDa. Immunocytochemistry revealed that tissues of the vascular cylinder contained abundant PRPs, particularly in the secondary cell walls of xylem elements and phloem fibers. PRPs were also found within the primary wall of the nodule endodermis and within Casparian strips of the vascular endodermis. Of symbiotic importance, PRPs were a prominent component of the infection thread matrix in newly infected root cells and in nodules. PRPs were also secreted by cells in the uninfected nodule parenchyma, where they were found occluding intercellular spaces outside the middle lamella. Despite structural conservation among members of this class of cell wall proteins, PRPs were targeted to distinct layers of the extracellular matrix dependent upon cell type, and may thus play separate roles in the biology of plant cells. The putative functions and the potential for interactions between PRPs and other wall polymers are discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine tetraacetate - GRP glycine-rich protein - PCR polymerase chain reaction - PGA polygalacturonic acid - PMSF phenylmethylsulfonyl fluoride - PRP proline-rich protein - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - Tris tris(hydroxylmethyl) aminomethane - Tween 20 polyoxyethylene sorbitan monolaurate Dedicated to the memory of Professor John G. Torrey