条形柄锈菌吸器特异效应蛋白Hasp68抑制寄主免疫并与小麦组织蛋白酶B互作

作为活体营养专性寄生真菌,条形柄锈菌（小麦条锈病）在侵染过程中通过形成吸器向寄主细胞释放效应蛋白,干扰寄主的防卫反应,促进其侵染与致病。因此,条形柄锈菌效应蛋白的鉴定与功能研究对揭示其毒性机理具有重要意义。本实验室前期完成了条形柄锈菌CYR31生理小种吸器转录组分析,从中鉴定得到一个吸器特异诱导表达分泌蛋白Hasp68,利用农杆菌侵染在烟草细胞中瞬时表达该基因,能够抑制小鼠促细胞凋亡蛋白Bax诱导的细胞程序性死亡,鉴定为条形柄锈菌候选效应蛋白。Hasp68基因全长318bp,编码105_aa,N-端包含20_aa的信号肽,无保守结构域。BlastX分析表明Hasp68为条形柄锈菌特有效应蛋白,在其他真菌中无同源蛋白,且在条形柄锈菌16个菌系中呈较低的序列多态性,表明其在条形柄锈菌的进化过程中相对保守。借助荧光假单胞菌EtHAn的三型分泌系统,在小麦细胞中过表达Hasp68能够抑制由非致病细菌引起的PTI（PAMP-triggered immunity）相关胼胝质的积累;同时,也能抑制小麦与无毒条形柄锈菌互作中ETI（effector-triggered immunity）相关的活性氧爆发和过敏性坏死反应,表明效应蛋白Hasp68具有抑制寄主免疫反应的功能。利用酵母双杂交系统筛选Hasp68在小麦中的互作蛋白,发现其与组织蛋白酶B（cathepsin B）TaCTSB互作,双分子荧光技术进一步验证二者在烟草细胞中共表达存在互作,初步揭示了效应蛋白Hasp68的互作靶标。     

Dimerization and protease resistance: New insight into the function of PR-1

The group 1 pathogenesis-related (PR-1) proteins have long been considered hallmarks of hypersensitive response/defense pathways in plants, but their biochemical functions are still obscure despite resolution of the NMR/X-ray structures of several PR-1-like proteins, including P14a (the prototype PR-1). We report here the characterization of two basic PR-1 proteins (PR-1-1 and PR-1-5) recently identified from hexaploid wheat (Triticum aestivum). Both proteins were expressed in Pichia pastoris as a single major species of ∼15 kDa. Sequence identity of the expressed PR-1 proteins was verified by MALDI-TOF/TOF analysis. Accumulation of the native PR-1-5 protein in pathogen-challenged wheat was confirmed by protein gel blot analysis. Low-temperature SDS-PAGE and yeast two-hybrid assays revealed that PR-1-1 exists primarily as a monomer whereas PR-1-5 forms homodimers. Both PR-1 proteins are resistant to proteases compared to bovine serum albumin, but PR-1-1 shows resistance mainly to subtilisin and protease K (serine proteases) whereas PR-1-5 shows resistance to subtilisin, protease K and papain (a cysteine protease). Site-specific mutations at the five putative active sites in the PR-1 domain all affected dimerization, with the mutations at Glu-72 and Glu-102 (in the PR-1-5 numeration) also diminishing protease resistance. Sequence analysis revealed that the Glu-72 and Glu-102 residues are located in motif-like sequences that are conserved in both PR-1 and the human apoptosis-related caspase proteins. These findings prompt us to examine the function of PR-1 for a role in protease-mediated programmed cell death pathways in plants.     

Tiller number is altered in the ascorbic acid-deficient rice suppressed for l-galactono-1,4-lactone dehydrogenase

The tiller of rice (Oryza sativa L.), which determines the panicle number per plant, is an important agronomic trait for grain production. Ascorbic acid (Asc) is a major plant antioxidant that serves many functions in plants. l-Galactono-1,4-lactone dehydrogenase (GLDH, EC 1.3.2.3) is an enzyme that catalyzes the last step of Asc biosynthesis in plants. Here we show that the GLDH-suppressed transgenic rices, GI-1 and GI-2, which have constitutively low (between 30% and 50%) leaf Asc content compared with the wild-type plants, exhibit a significantly reduced tiller number. Moreover, lower growth rate and plant height were observed in the Asc-deficient plants relative to the trait values of the wild-type plants at different tillering stages. Further examination showed that the deficiency of Asc resulted in a higher lipid peroxidation, a loss of chlorophyll, a loss of carotenoids, and a lower rate of CO₂ assimilation. In addition, the level of abscisic acid was higher in GI-1 plants, while the level of jasmonic acid was higher in GI-1 and GI-2 plants at different tillering stages. The results we presented here indicated that Asc deficiency was likely responsible for the promotion of premature senescence, which was accompanied by a marked decrease in photosynthesis. These observations support the conclusion that the deficiency of Asc alters the tiller number in the GLDH-suppressed transgenics through promoting premature senescence and changing phytohormones related to senescence.     

Leishmania donovani: dyskinetoplastid cells survive and proliferate in the presence of pyruvate and uridine but do not undergo apoptosis after treatment with camptothecin

We have shown that treatment with luteolin in leishmanial cells causes loss of mt-DNA and induces apoptosis through mitochondria dependent pathway [Sen, N., Das, B.B., Ganguly, A., Banerjee, B., Sen, T., Majumder, H.K., 2006. Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells. Experimental Parasitology, in press]. Here, we report that mitochondrial DNA depleted leishmanial cells require exogenous sources of pyruvate and uridine to survive and proliferate. The presence of pyruvate and uridine in a growing media help them to produce sufficient amount of glycolytic ATP to maintain the mitochondrial membrane potential in the absence of their functional ETC. Treatment of wild type cells with CPT causes generation of ROS that leads to apoptosis. But unlike the normal cells ROS was not generated in these mt-DNA depleted cells after treatment with CPT. Taken together we have shown for the first time that dyskinetoplastid cells are auxotrophic for pyruvate and uridine and apoptosis cannot be induced in these cells in the presence of CPT. Therefore, the presence of mitochondrial DNA is absolutely necessary for the cytotoxicity of CPT in kinetoplastid parasites.     

The peptaibol alamethicin induces an rRNA-cleavage-associated death in Arabidopsis thaliana

The plant-metabolic response to amphipathic peptides produced by the soil fungi of the genus Trichoderma remains largely unknown. The present investigation was undertaken to examine the death process in alamethicin-treated Arabidopsis thaliana plantlets. The rapid death triggered by alamethicin (at 50 microM) was shown to be associated with protein-synthesis arrest and with specific cleavage of 18S and 25S ribosomal RNA. The use of an inhibitor of nitric oxide (NO) synthases and of an NO scavenger suggested that rRNA cleavage was suppressed by NO. Experiments conducted with a synthetic alamethicin analogue, in which all alpha-aminoisobutyric acid (Aib) residues have been replaced by leucine moieties, showed that the non-coded residues are essential for the ability of the peptaibol to induce rRNA cleavage in Arabidopsis. Our data indicate that further investigations on the mode of action of alamethicin in planta could be of great interest to study the death-signaling pathway associated with rRNA degradation in plants.     

Infection by a Hematodinium-like parasitic dinoflagellate causes Pink Crab Disease (PCD) in the edible crab Cancer pagurus

The edible crab (Cancer pagurus) supports a large and valuable fishery in UK waters. Much of the catch is transported live to continental Europe in specially designed live-well ('vivier') vehicles. During the winter of 2000/2001, many trap-caught crabs from Guernsey, Channel Islands, UK, were reportedly moribund and pink in colour. These crabs generally died before and during vivier transportation. We provide histological, immunological, and molecular evidence that this condition is associated with infection by a Hematodinium-like dinoflagellate parasite similar to that previously reported in C. pagurus and to an infection causing seasonal mass mortalities of the Norway lobster (Nephrops norvegicus). Pathologically, every altered host bore the infection, which was characterised by very large numbers of plasmodial and vegetative stages in the haemolymph and depletion of reserve cells in the hepatopancreas. Due to the hyperpigmentation of the carapace and appendages, we have called this infection 'Pink Crab Disease' (PCD). Similar Hematodinium infections cause 'Bitter Crab Disease' in tanner and snow crabs, which has had a negative effect on their marketability. At present, little is known about the seasonality, transmission, and market impact of this infection in C. pagurus.     

植物细胞程序死亡的机理及其与发育的关系

细胞程序死亡（ＰＣＤ）是在植物体发育过程中普遍存在的,在发育的特定阶段发生的自然的细胞死亡过程,这一死亡过程是由某些特定基因编码的“死亡程序”控制的。ＰＣＤ的细胞分化的最后阶段。细胞分化的临界期就牌死亡程序执行中的某个阶段。ＰＣＤ包含启动期和清除期三个阶段,其间ＣＡＳＰＡＳＥ家族起着重要作用。ＰＣＤ在细胞和组织的平衡、特化,以及组织分化、器官建成和对病原体的反应等植物发育过程中起着重要作用。ＰＣＤ     

Changes in cell structure during the formation of root aerenchyma inSAGITTARIA LANCIFOLIA (Alismataceae)

In many wetland species, root aerenchyma is produced by the predictable collapse of root cortex cells, indicating a programmed cell death (PCD). The objective of this study was to characterize the cellular changes that accompany this PCD in the marsh species Sagittaria lancifolia. Structural changes in membranes and organelles were examined during development of root cortex cells to compare with previous examples of PCD. The organization of cortical microtubule (CMT) arrays in root cells from S. lancifolia was also evaluated as a possible predictor of cell lysis. Nuclear fragmentation and condensation were the earliest changes observed in cells undergoing lysis. Breakdown of the tonoplast and other organelles and disruption of the plasma membrane followed. After loss of cytoplasm, cells collapsed to form gas spaces. These results were compared to collapse of root cortical cells of Zea mays and Oryza sativa during aerenchyma development. Changes in the appearance of the cytoplasm of all three species were similar at later stages of aerenchyma development. The relative timing of disintegration of the tonoplast and middle lamella appeared to differ among the three species. Changes in the organization of CMT arrays did not appear to be a predictor of PCD in S. lancifolia. Aerenchyma production in plants involves a type of PCD that is morphologically distinct from PCD described from many animals.