杧果Whirly基因鉴定及其在病菌侵染过程中的表达分析

Whirly家族是一类植物特异的转录因子,无论在细胞核还是在细胞器内都有着广泛而复杂的生物学功能。该研究以杧果全基因组数据为基础,采用生物信息学的方法对杧果Whirly家族基因进行序列分析,并通过qRT-PCR技术分析杧果胶孢炭疽菌(Colletotrichum gloeosporioides,Cg)和细菌性黑斑病菌(Xanthomonas campestris pv.mangiferaeindicae,Xcm)侵染过程中杧果Whirly家族基因的相对表达量。结果表明:(1)从杧果基因组中鉴定了3个Whirly基因家族成员,分别命名为MiWHY1、MiWHY2和MiWHY3。(2)杧果Whirly蛋白均为不稳定亲水碱性蛋白;系统发育树显示,杧果Whirly基因与木薯、毛果杨、番茄Whirly基因亲缘关系最近,杧果与木薯、毛果杨、番茄Whirly家族共有1个高度保守的基序Motif1;杧果Whirly家族均含有Whirly超家族保守域,主要结构元件为无规则卷曲和α-螺旋;保守结构域的四聚体结构与马铃薯Whirly蛋白四聚体结构具有高度相似性。(3)qRT-PCR结果显示,Cg侵染过程中,杧果Whirly基因的相对表达量均显著上调;Xcm侵染12 h时,MiWHY1和MiWHY3相对表达量显著上调,初步确定杧果Whirly基因表达响应胶孢炭疽菌和细菌性黑斑病菌的侵染。研究认为,杧果中有3个Whirly基因家族成员,病菌侵染过程中可激发其表达活性,为后续研究杧果Whirly基因家族成员的功能和机制奠定基础。     

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer-tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.     

Cellulose binding domains of a Phytophthora cell wall protein are novel pathogen-associated molecular patterns

The cellulose binding elicitor lectin (CBEL) from Phytophthora parasitica nicotianae contains two cellulose binding domains (CBDs) belonging to the Carbohydrate Binding Module1 family, which is found almost exclusively in fungi. The mechanism by which CBEL is perceived by the host plant remains unknown. The role of CBDs in eliciting activity was investigated using modified versions of the protein produced in Escherichia coli or synthesized in planta through the potato virus X expression system. Recombinant CBEL produced by E. coli elicited necrotic lesions and defense gene expression when injected into tobacco (Nicotiana tabacum) leaves. CBEL production in planta induced necrosis. Site-directed mutagenesis on aromatic amino acid residues located within the CBDs as well as leaf infiltration assays using mutated and truncated recombinant proteins confirmed the importance of intact CBDs to induce defense responses. Tobacco and Arabidopsis thaliana leaf infiltration assays using synthetic peptides showed that the CBDs of CBEL are essential and sufficient to stimulate defense responses. Moreover, CBEL elicits a transient variation of cytosolic calcium levels in tobacco cells but not in protoplasts. These results define CBDs as a novel class of molecular patterns in oomycetes that are targeted by the innate immune system of plants and might act through interaction with the cell wall.     

植物中逆境反应相关的WRKY转录因子研究进展

WRKY转录因子是植物体内一类比较大的转录因子家族,它在植物的生长发育以及抗逆境反应中起着非常重要的作用。本文综述了WRKY转录因子在植物应对冻害、干旱、盐害等非生物胁迫与病原菌、虫害等生物胁迫反应中的重要调控功能,并概括了WRKY转录因子在调控这些逆境反应中的机制。     

The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis

Copines are calcium-dependent membrane-binding proteins that are highly conserved among protozoa, plants, nematodes and mammals. Although they are implicated in membrane trafficking and signal transduction, the functions of these proteins are not well understood. The Arabidopsis copine gene BON1/CPN1 was previously shown to negatively regulate a disease resistance (R) gene SNC1. Here we report that in Arabidopsis, as in other organisms, there is a family of copine genes, BON1, 2 and 3. Using double and triple mutant combinations we show that these three copine genes have overlapping functions essential for the viability of plants. The loss of function of BON1 combined with that of BON2 or BON3 leads to extensive cell death phenotypes resembling the hypersensitive response (HR) in defense responses. The resulting lethality can be suppressed by mutations in PAD4 or EDS1 which are required for R gene signaling and cell death control. Accession-dependent phenotypes of the mutant combinations suggest that the BON/CPN genes may together repress several R genes other than SNC1. Moreover, the mutant combinations exhibit developmental defects when R-gene-mediated defense responses are largely suppressed in pad4 and eds1 mutants. Thus, the copine family in Arabidopsis may have effects in promoting growth and development in addition to repressing cell death, and these two processes might be intricately intertwined.     

Single-stranded DNA binding factor AtWHY1 modulates telomere length homeostasis in Arabidopsis

Telomere homeostasis, a process that is essential for the maintenance of chromosome integrity, is regulated by telomerase and a collection of associated proteins. By mass spectrometry we have identified a new telomeric protein encoded by the AtWHY1 (Arabidopsis thaliana Whirly 1) gene in Arabidopsis. AtWHY1 specifically binds the single-stranded plant telomeric DNA sequences, but not double-stranded telomeric DNA. To gain insights into the function of AtWHY1 in telomere biogenesis, we have identified two Arabidopsis lines harboring T-DNA insertions in AtWHY1. These lines exhibit neither growth nor developmental defects. However, AtWHY1-deficient plants show a steady increase in the length of telomere tracts over generations. This telomere elongation is correlated with a significant increase in telomerase activity. On the contrary, transgenic plants expressing AtWHY1 show a decreased telomerase activity and shortened telomeres. The evidence presented here indicates that AtWHY1 is a new family of telomere end-binding proteins that plays a role in regulating telomere-length homeostasis in Arabidopsis.     

Expression Profiles of Arabidopsis thaliana VQ Gene Family in Defense Related Hormones Treatments

Several VQ proteins are recently identified as WRKY factors interacting partners in Arabidopsis and involved in regulating physiological processes. Searching of genomic databases found that VQ gene family is specific to land plants and these VQ genes encode proteins characteristic of a conserved VQ motif. It consists of 34 representatives in Arabidopsis and can be divided into two groups based on the similarity of the amino acid sequences. To understand the functions of Arabidopsis VQ proteins, we examined the expression profiles of AtVQ genes in various defense related hormones treatments. qRT PCR analysis revealed that a majority of them were differently regulated in response to salicylic acid (SA), methyl jasmonate (MeJA), or 1 aminocyclopropane 1 carboxylate (ACC). And some members are induced by two of these three hormones. Moreover, four members (AtVQ3, AtVQ18, AtVQ23 and AtVQ24) are induced by SA, MeJA and ACC simultaneity. However, there is only one AtVQ gene (AtVQ27) is up regulated after spraying of abscisic acid (ABA). These results suggest that Arabidopsis VQ genes may be involved in plant defense responses.     

The HopZ family of Pseudomonas syringae type III effectors require myristoylation for virulence and avirulence functions in Arabidopsis thaliana

Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a(PsyA2), HopZ1b(PgyUnB647), HopZ1c(PmaE54326), HopZ2(Ppi895A) and HopZ3(PsyB728a). HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.     

拟南芥VQ基因家族响应抗性相关激素表达谱分析

近年来与WRKY转录因子相互作用的VQ-motif蛋白逐渐引起广泛关注.它是一类植物特异性蛋白,目前在拟南芥(Arabidopsis thaliana)中已鉴定到34个成员,据其所编码氨基酸的序列相似性构建系统进化树,这34个成员聚集成两大簇.实时定量荧光PCR分析表明在SA(水杨酸),MeJA(茉莉酸甲酯),ACC(乙烯前体),ABA(脱落酸)处理下AtVQ基因家族中有多个成员分别受SA,JA,ET高倍诱导,部分成员受SA,JA,ET中两种激素诱导,其中AtVQ3,AtVQ18,AtVQ23和AtVQ24同时受SA,JA,ET诱导,仅AtVQ27受ABA诱导,推测拟南芥VQ家族成员在抗病方面起作用.     

Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.