A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14

Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.     

Isolation and characterization of a gene encoding a drought-induced cysteine protease in tomato (Lycopersicon esculentum).

In a previous study, a 65 kDa protein, TDI-65, was found to be accumulated in the leaves of drought-stressed tomato (Lycopersicon esculentum cv. Starfire) plants. The protein level returns to control level when the drought-stressed plants are rewatered. Antibodies raised against the purified protein were used to elucidate the subcellular localization of the protein. The protein was found to be mainly localized in the nuclei and chloroplasts of drought-stressed leaf cells. To identify the nature of the protein, a cDNA library was constructed and screened by the purified anti-TDI-65 antibody. A cDNA clone designated tdi-65 was isolated and characterized. The deduced amino acid sequences of tdi-65 protein has extensive homology with known cysteine proteases such as actinidin and papain. Northern blot analysis revealed that tdi-65 mRNA is 10-fold higher in drought-stressed plants as compared to control and rewatered plants. Similar results were observed in the tomato cultivar Ailsa and its near isogenic abscisic acid (ABA)-deficient mutant line, flacca, suggesting that the gene does not require ABA for its expression under drought conditions. Based on the previous immunolocalization findings we suggest that tdi-65 encoded cysteine protease functions in relation to drought-induced senescence and programmed cell death.     

Isolation of the protease component of maize cysteine protease-cystatin complex: release of cystatin is not crucial for the activation of the cysteine protease

The maize cysteine protease complex, which required SDS for its activation in vitro, is a 179 kDa trimeric complex (P-I)3 of a cysteine protease (P) [EC 3.4.22] and a cysteine protease inhibitor (I), cystatin [Yamada et al. (1998) Plant Cell Physiol. 39: 106, Yamada et al. (2000) Plant Cell Physiol. 41: 185]. Here, we show the mechanism of the SDS-dependent activation of the trimeric (P-I) complex and stabilization of the activated protease by its specific substrates. The cystatin-free cysteine protease isolated by preparative SDS-PAGE was still specifically activated by SDS, and its profile of SDS-dependency was exactly the same as that of the trimeric (P-I) complex. It is, therefore, evident that an SDS-dependent conformational change of the protease itself, rather than the release of cystatin from the complex, is crucial for the activation. Pre-treatment analysis with SDS revealed that SDS was required for the initiation of the activation of the trimeric (P-I) complex. Furthermore, we found that once the protease was activated, if there was no substrate, it was rapidly inactivated under optimum conditions of proteolysis, and showed that such inactivation was not due to autolysis of the protease. In contrast, addition of specific substrates prevented the inactivation, and thus we presumed that the activity of the cysteine protease is regulated by both activation by conformational change and rapid inactivation after consumption of substrates.     

侧孢芽孢杆菌Bl13对番茄早疫病防治效果及机制

以对番茄早疫病原菌有良好拮抗效果的侧孢芽孢杆菌Bl13为研究对象,采用盆栽试验,通过测定番茄株高、茎粗、番茄早疫病病情指数、叶片内防御酶活性以及根区土壤微生物多样性、微生物群落结构组成等指标,探究侧孢芽孢杆菌Bl13防治番茄早疫病的效果及机制。结果表明: 接种Bl13可显著降低番茄早疫病的病情指数,提高叶片内多酚氧化酶(PPO)、过氧化物酶(POD)等防御酶活性,降低病害对植物地上部分及根系生长发育的影响。同时,改善番茄根区土壤微生物群落结构,使芽孢杆菌属、假单胞菌属等常见有益菌属相对丰度显著提高,油壶菌属、血赤壳属相对丰度显著降低。侧孢芽孢杆菌Bl13可通过提高番茄叶片内防御酶活性并增加根区中有益微生物的数量来增强植物对番茄早疫病的抗性,从而实现对番茄早疫病的防治。     

Diversity,abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation

A novel class of cysteine phytase showing ability to degrade phytate has recently been isolated from rumen bacteria. To expand our knowledge of this enzyme class, a total of 101 distinct cysteine phytase gene fragments were identified from the ruminal genomic DNA of Bore goats and Holstein cows, and most of them shared low identities (< 50%) with known sequences. By phylogenetic analysis, these sequences were separated into three clusters that showed substantial diversity. The two most abundant cysteine phytase genes of goat rumens were cloned and their protein products were characterized. Four findings were revealed based on our results. (i) Compared with soil and water environment, where β‐propeller phytase is the most important phytate‐degrading enzyme, cysteine phytase is the major phytate‐degrading enzyme in the anaerobic ruminal environment. (ii) Cysteine phytase fragments in the rumen contents of goat and cow have the same diversity profile, although most of the sequences and their abundance differ in the two species. (iii) Each species has their respective high‐abundance genes, which may play major roles for phytate degradation. (iv) Compared with previously reported cysteine phytases that have pH optimum at 4.5, the pH optima of the two most abundant secreted goat cysteine phytases are 6.5 and 6.0, which are within the pH range found in the rumens. This study provides valuable information about the diversity, abundance and enzymatic properties of the ruminal cysteine phytases and emphasizes the important role(s) of these cysteine phytases probably in the terrestrial cycle of phosphorus.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

家蝇半胱氨酸蛋白酶抑制剂基因MdCPI的克隆、表达及活性检测

半胱氨酸蛋白酶抑制剂是具有抑制半胱氨酸蛋白酶活性的一类蛋白超家族。本研究根据EST序列信息,通过RACE技术克隆得到1条家蝇Musca domestica半胱氨酸蛋白酶抑制剂基因MdCPI,该基因含有1个357 bp开放阅读框,编码118个氨基酸残基,推导的多肽N端17个氨基酸残基为信号肽序列。同源分析表明,MdCPI 氨基酸序列与红尾肉蝇Sarcophaga crassipalpis的CPI相似性最高（identity=51%）。以邻接法（NJ）构建的系统树表明,家蝇与其他双翅目昆虫CPI起源于共同的祖先,属于I25A型蛋白家族。为了解家蝇CPI对半胱氨酸蛋白酶的抑制活性,构建pET-17b-MdCPI表达载体,并转入大肠杆菌Escherichia coli BL21（DE3）进行重组表达。研究发现1 μg重组家蝇CPI能够抑制约14 μg木瓜蛋白酶的水解活性。结果表明MdCPI确属CPI家族,可能同其他家族成员具有相似的功能,参与免疫及生理调控。这些结果为研究MdCPI在家蝇体内作用机制奠定了基础。     

Isolation and characterization of a cysteine protease of freesia corms

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms.     

Cloning and characterization of tomato leaf senescence-related cDNAs

Senescence-related cDNA clones designated SENU1, 4, 5 (senescence up-regulated) and SEND32, 33, 34, 35 and 36 (senescence down-regulated) isolated from a tomato leaf cDNA library [9] were characterized. Southern analysis showed that SEND32 is encoded by a single-copy gene while SEND33, 34, 35, 36 and SENU1 and SENU5 are members of small gene families. DNA and protein database searches revealed that SEND32, SEND35, SENU1 and SENU5 are novel cDNAs of unknown function. SEND33 encodes ferredoxin, SEND34 encodes a photosystem II 10 kDa polypeptide and SEND36 encodes catalase. The SENU4 sequence is identical to the P6 tomato protein previously reported to be pathogenesis-related [46]. The mRNA levels of SENU1, 4 and 5 increased during leaf senescence and SENU1 and SENU5 were also expressed at high levels during leaf development and in other plant organs. The SENU4 mRNA was associated more specifically with leaf senescence, although low expression was also detected in green fruit. The mRNAs for all SEND clones decreased during tomato leaf development and senescence and all except SEND32 were expressed at low levels in other plant organs. The accumulation of mRNA homologous to SENU4 and the decrease in abundance of SEND32 provide good molecular markers for leaf senescence.     

Expression and characterization of cathepsin L-like cysteine protease from Philasterides dicentrarchi

Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.     

Cloning, expression, purification, and characterization of Streptococcus pneumoniae IgA1 protease

The IgA1 protease of Streptococcus pneumoniae is a Zn-metalloproteinase of 1964 amino acids that specifically cleaves the hinge region of IgA1, the predominant class of immunoglobulin present on mucosal membranes. This protease is associated to the bacterial cell surface via an N-terminal membrane anchor. Following proteolysis it is released in several forms of different molecular weight. Here, we describe the cloning, expression, and characterization of the enzymatic activity and immunogenicity of three fragments of IgA1 protease, including a large one lacking only the 103 N-terminal amino acids that constitute a typical prokaryotic signal sequence. Further, a proteolytically inactive mutant was generated by replacement of the glutamate residue with an alanine residue in the active site motif HExxH (1605-1609). This is the first report of recombinant active forms of S. pneumoniae IgA1 protease, which open the possibility of identifying specific inhibitors that could interfere with the mucosal colonization by pneumococcus. Moreover the inactive mutant could be considered as a candidate vaccine component.     

Reduced sensitivity of tomato early blight pathogen (Alternaria solani) isolates to protectant fungicides,and implication on disease control

The sensitivity of Alternaria solani isolates to the fungicides mancozeb and chlorothalonil was evaluated, to determine if inadequate disease management by these fungicides could be attributed to reduced sensitivity of A. solani isolates to these fungicides. The sensitivity of 60 isolates of A. solani was assessed using the inhibition of radial mycelial growth (RG) method, using fungicide concentrations of 0, 1.0, 10, 100, 500 and 1000 μg a.i ml^?1 medium. EC₅₀ was calculated for each isolate and fungicide combination. The EC₅₀ values of different A. solani isolates to mancozeb ranged from 9.05 to 712.65 μg ml^?1. EC₅₀ values of different isolates to chlorothalonil ranged from 4.25 to 849.4 μg ml^?1. The percentage of isolates with reduced sensitivity was 46.7 and 53.3% for mancozeb and chlorothalonil, respectively. Results of the in vivo tests demonstrated decline in disease control by the two fungicides with the reduced-sensitivity isolates compared to the sensitive ones.     

地表球囊霉诱发番茄抗早疫病的机理

丛枝菌根可以改善植物营养状况,提高宿主植物的抗病性.本文研究了番茄幼苗预先接种丛枝菌根真菌(AMF)地表球囊霉后对番茄植株保护酶活性和防御反应基因表达,以及对番茄早疫病抗性的影响.结果表明:被AMF侵染的番茄植株在接种早疫病病原菌茄链格孢菌后,其叶片内的超氧化物歧化酶(SOD)和过氧化物酶(POD)活性迅速提高.其中SOD酶活性在接种后18h达到最高,比只接种地表球囊霉(G)、茄链格孢菌(A)以及未接种AMF和病原菌的对照(CK)分别高28.6％、79.2％和82.8％;POD酶活性在接种后65 h达到最高,分别比G、A处理和CK高762％、18.3％和1710％.经荧光定量PCR检测表明,AMF侵染后的番茄植株再接种病原菌,其叶片中PR1(病程相关蛋白基因)、PR2(β-1,3-葡聚糖酶基因)和PR3(几丁质酶基因)基因的最高转录水平达到CK的9.67、8.54和13.4倍.与CK相比,先接种地表球囊霉再接种茄链格孢菌的番茄植株(GA)的早疫病发病率和病情指数分别降低了36.3％和61.4％.预先接种AMF的番茄植株在遇到病原菌袭击时诱导的防御反应强而迅速,诱发(priming)可能是菌根真菌提高宿主植物抗病性的重要机制.     

The effect of early blight disease caused by Alternaria solani on shoot growth of young tomato plants

The effect of increasing spore concentration of Alternaria solani (Early blight disease) on the shoot growth of young tomato plants was analysed. Changes in growth were related to the severity of infection which increased with increasing inoculum. Leaf production was not affected but dry weights and especially leaf expansion were decreased. The effective leaf areas of the five inoculated leaves (L1-L5 numbered from the plant base) were drastically decreased by expanding necrotic lesions and, to a lesser extent, by premature leaf fall. Healthy leaves expanding soon after inoculation (L6, L7) were markedly affected by the disease on the lower leaves and had decreased specific leaf areas (ratio of leaf area to leaf dry weight) but later formed (from L8) leaves were less affected and had greater specific leaf areas than equivalent leaves on uninoculated plants.     

Cloning and characterization of a mammalian prenyl protein-specific protease

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.     

Development of alpha-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1' residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3A crystallographic structure of cruzain bound with one of the alpha-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization.