Characterization of protein kinase PsSRPKL,a novel pathogenicity factor in the wheat stripe rust fungus

As in other eukaryotes, protein kinases (PKs) are generally evolutionarily conserved and play major regulatory roles in plant pathogenic fungi. Many PKs have been proven to be important for pathogenesis in model fungal plant pathogens, but little is currently known about their roles in the pathogenesis of cereal rust fungi, devastating pathogens in agriculture worldwide. Here, we report on an in planta highly induced PK gene PsSRPKL from the wheat stripe rust fungus Puccinia striiformis f. sp. tritici (Pst), one of the most important cereal rust fungi. PsSRPKL belongs to a group of PKs that are evolutionarily specific to cereal rust fungi. It shows a high level of intraspecies polymorphism in the kinase domains and directed green fluorescent protein chimers to plant nuclei. Overexpression of PsSRPKL in fission yeast induces aberrant cell morphology and a decreased resistance to environmental stresses. Most importantly, PsSRPKL is proven to be an important pathogenicity factor responsible for fungal growth and responses to environmental stresses, therefore contributing significantly to Pst virulence in wheat. We hypothesize that cereal rust fungi have developed specific PKs as pathogenicity factors for adaptation to their host species during evolution. Thus, our findings provide significant insights into pathogenicity and virulence evolution in cereal rust fungi.     

小麦与条锈菌互作中过氧化物酶的细胞化学研究

采用细胞化学方法对小麦与条锈菌互作过程中过氧化物酶的分布及其活性大小进行了研究,结果表明：过氧化物酶主要分布于细胞壁和细胞间隙中;在未行接种的小麦叶片中,抗病品种和感病品种的过氧化物酶活性均比较低;条锈菌侵染后,诱导抗、感病品种叶片中的过氧化物酶活性升高,且抗病品种升高的幅度明显大于感病品种;感病品种中过氧化物酶活性在侵染位点附近细胞壁上表现升高,而抗病品种中该酶的活性在侵染点细胞以及远离侵染点的叶肉细胞的细胞壁和细胞间隙中均显著升高。高活性的过氧化物酶是小麦抗条锈性的生化标记和重要机制之一。     

条形柄锈菌(小麦条锈病菌)侵染结构体外形成的观察

通过荧光显微镜和扫描电镜分别对条形柄锈菌夏孢子在寄主植物-小麦叶表和非寄主植物-水稻叶表以及小麦穗部和茎秆上的萌发过程进行了观察。结果发现,夏孢子在小麦叶片体表萌发产生芽管后,可依次分化形成气孔下囊、初生菌丝与吸器母细胞;在小麦颖片、稃片及茎秆部位表面,同样可观察到病菌在体外分化形成吸器母细胞;并且在水稻叶片上也观察到病菌侵染结构存在体外分化现象。经荧光染色发现,条形柄锈菌在体外与在小麦组织中形成的侵染结构没有明显的差别。观察结果可为条形柄锈菌侵染结构的离体诱导与调控机理研究提供依据。     

Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction

Insulin signaling involves a dynamic cascade of protein tyrosine phosphorylation and dephosphorylation. Most of our understanding of this process comes from studies focusing on tyrosine kinases, which are signal activators. Our knowledge of the role of protein-tyrosine phosphatases (PTPases), signal attenuators, in regulating insulin signal transduction remains rather limited. Protein-tyrosine phosphatase 1B (PTP-1B), the prototypical PTPase, is ubiquitously and abundantly expressed. Work from several laboratories, including our own, has implicated PTP-1B as a negative regulator of insulin action and as a potentially important mediator in the pathogenesis of insulin-resistance and non-insulin dependent diabetes mellitus (NIDDM).     

Bfl-1/A1 acts as a negative regulator of autophagy in mycobacteria infected macrophages

Expression of Bcl-2 family protein, Bfl-1/A1 has been found to differ considerably amongst macrophages infected with virulent Mycobacterium tuberculosis H37Rv or with avirulent M. tuberculosis H37Ra. Present work was undertaken to deduce the significance of differential expression of Bfl-1/A1 in the outcome of mycobacterial infection. We have studied the role of Bfl-1/A1 particularly in autophagy formation in tubercle bacilli infected cells since autophagy has been recognized as a component of innate immunity against pathogenic mycobacteria. First, we have confirmed that upon infection virulent strain H37Rv retain Bfl-1/A1 for longer period and impose autophagosome maturation block within infected cells as evident from confocal microscopy. Moreover, down regulation of Bfl-1/A1 by siRNA induced autophagy formation and reduced bacterial growth. Furthermore, even the avirulent strain H37Ra resist autophagosome maturation and survive if the cellular level of Bfl-1 is maintained in THP-1 cells by stable transfection (Bfl-1 overexpressing cells). No noteworthy difference in mTOR expression was observed between normal THP-1 and Bfl-1 overexpressing THP-1 cells infected with either strain of mycobacteria. Interestingly, we found that not only mTOR but also Bfl-1/A1 is involved in rapamycin induced autophagy in mycobacteria infected macrophages. We have found that Bfl-1 physically interacts with Beclin 1 in Bfl-1 overexpressing THP-1 as well as in H37Rv infected THP-1 cells as they co-precipitated. Taken together, our results clearly demonstrated that Bfl-1/A1 negatively regulates autophagy and expression of Bfl-1/A1 in H37Rv infected macrophages provides the bacteria a survival strategy to overcome host defense.     

The type-A response regulator,ARR15, acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana

The Arabidopsis thaliana AHK4 histidine kinase (also known as CRE1 or WOL) acts as a cytokinin signal transducer, presumably, in concert with downstream components, such as histidine-containing phosphotransfer factors (AHPs) and response regulators (ARRs), through the histidine-to-aspartate (His-->Asp) phosphorelay. Among 10 members of the type-A ARR family, the cytokinin-induced expression of ARR15 in roots is selectively impaired in the cre1-1 mutant, which carries a mutation in the AHK4 gene, suggesting a link between this type-A response regulator and the AHK4-mediated cytokinin signal transduction in roots. To address this issue further, we characterized a T-DNA insertion mutant of ARR15, and also constructed transgenic lines (referred to as ARR15-ox) that overexpress the ARR15 gene in a manner independent of cytokinin. While the T-DNA insertion mutant (arr15-1) showed no apparent phenotype, the cytokinin-independent overexpression of ARR15 in ARR15-ox plants resulted in a reduced sensitivity toward exogenously applied cytokinin, not only in elongation of roots in plants, but also in green callus formation (or shoot formation) in explants. Cytokinin-induced expressions of certain type-A ARRs were also down-regulated in ARR15-ox plants. These results support the view that ARR15 acts as a repressor that mediates a negative feedback loop in the cytokinin and AHK4-mediated His-->Asp phosphorelay.     

QTL characterization of resistance to leaf rust and stripe rust in the spring wheat line Francolin#1

Growing resistant wheat varieties is a key method of controlling two important wheat diseases, leaf rust and stripe rust. We analyzed quantitative trait loci (QTL) to investigate adult plant resistance (APR) to these rusts, using 141 F₅ RILs derived from the cross ‘Avocet-YrA/Francolin#1’. Phenotyping of leaf rust resistance was conducted during two seasons at Ciudad Obregon, Mexico, whereas stripe rust was evaluated for two seasons in Toluca, Mexico, and one season in Chengdu, China. The genetic map was constructed with 581 markers, including diversity arrays technology and simple sequence repeat. Significant loci for reducing leaf rust severity were designated QLr.cim-1BL, QLr.cim-3BS.1, QLr.cim-3DC, and QLr.cim-7DS. The six QTL that reduced stripe rust severity were designated QYr.cim-1BL, QYr.cim-2BS, QYr.cim-2DS, QYr.cim-3BS.2, QYr.cim-5AL, and QYr.cim-6AL. All loci were conferred by Francolin#1, with the exception of QYr.cim-2DS, QYr.cim-5AL, and QYr.cim-6AL, which were derived from Avocet-YrA. Closely linked markers indicated that the 1BL locus was the pleiotropic APR gene Lr46/Yr29. QYr.cim-2BS was a seedling resistance gene designated as YrF that conferred intermediate seedling reactions and moderate resistance at the adult plant stage in both Mexican and Chinese environments. Significant additive interactions were detected between the six QTL for stripe rust, but not between the four QTL for leaf rust. Furthermore, we detected two new APR loci for leaf rust in common wheat: QLr.cim-3BS.1 and QLr.cim-7DS.     

SHORT HYPOCOTYL IN WHITE LIGHT1, a serine-arginine-aspartate-rich protein in Arabidopsis, acts as a negative regulator of photomorphogenic growth

Light is an important factor for plant growth and development. We have identified and functionally characterized a regulatory gene SHORT HYPOCOTYL IN WHITE LIGHT1 (SHW1) involved in Arabidopsis (Arabidopsis thaliana) seedling development. SHW1 encodes a unique serine-arginine-aspartate-rich protein, which is constitutively localized in the nucleus of hypocotyl cells. Transgenic analyses have revealed that the expression of SHW1 is developmentally regulated and is closely associated with the photosynthetically active tissues. Genetic and molecular analyses suggest that SHW1 acts as a negative regulator of light-mediated inhibition of hypocotyl elongation, however, plays a positive regulatory role in light-regulated gene expression. The shw1 mutants also display shorter hypocotyl in dark, and analyses of shw1 cop1 double mutants reveal that SHW1 acts nonredundantly with COP1 to control hypocotyl elongation in the darkness. Taken together, this study provides evidences that SHW1 is a regulatory protein that is functionally interrelated to COP1 and plays dual but opposite regulatory roles in photomorphogenesis.     

AINTEGUMENTA promotes petal identity and acts as a negative regulator of AGAMOUS

The Arabidopsis AINTEGUMENTA (ANT) gene has been shown previously to be involved in ovule development and in the initiation and growth of floral organs. Here, we show that ANT acts in additional processes during flower development, including repression of AGAMOUS (AG) in second whorl cells, promotion of petal epidermal cell identity, and gynoecium development. Analyses of ap2-1 ant-6 double mutants reveal that ANT acts redundantly with AP2 to repress AG in second whorl cells. The abaxial surface of ant petals contains features such as stomata and elongated, interdigitated cells that are not present on wild-type petals. The loss of petal identity in these second whorl cells does not result from ectopic AG expression, suggesting that ANT acts in a pathway promoting petal cell identity that is independent of its role in repression of AG. These data suggest that ANT may function as a class A gene.     

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.