Advances and prospects in potato virology with special reference to virus resistance

Knowledge of the nucleotide sequences in the genomic nucleic acid of several potato viruses has enabled the open reading frames to be identified. These open reading frames are expressed by a variety of strategies, to produce proteins with functions in virus nucleic acid replication, virus particle production, cell-to-cell transport of virus and virus transmission by vectors. The activity of such proteins depends on their interactions with other viral or non-viral materials.Several other biological properties of plant viruses can also be related to individual viral gene products. For example, in plants co-infected with a specific pair of unrelated viruses, one virus can benefit from an ability to use the gene product of the second virus in replication, cell-to-cell transport or transmission by vectors. Similarly, different host resistance genes are targeted against viral replicase, movement protein or coat protein. Thus it is becoming possible to relate gene-for-gene (or more accurately, viral gene domain-host gene) interactions to events at the molecular level. Genetically engineered resistance to plant viruses likewise can be targeted against individual viral genes, and probably also against viral regulatory sequences. Such transgenic resistance seems likely to be as durable as conventional host resistance but durability should be improved by producing plants with combinations of resistances of different kinds, either conventional or genetically engineered, or both.     

马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性

为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。     

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

Phytoplasma: ecology and genomic diversity

ABSTRACT The recent development of molecular-based probes such as mono- and polyclonal antibodies, cloned phytoplasma DNA fragments, and phytoplasma-specific primers for polymerase chain reaction (PCR) has allowed for advances in detection and identification of uncultured phytoplasmas (formerly called mycoplasma-like organisms). Comprehensive phylogenetic studies based on analysis of 16S ribosomal RNA (rRNA) or both 16S rRNA and ribosomal protein gene operon sequences established the phylogenetic position of phytoplasmas as members of the class Mollicutes, and the revealed phylogenetic interrelationships among phytoplasmas formed a basis for their classification. Based on restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rRNA gene sequences, phytoplasmas are currently classified into 14 groups and 38 subgroups that are consistent with groups delineated based on phylogenetic analysis using parsimony of 16S rRNA gene sequences. In the past decades, numerous phyto-plasma strains associated with plants and insect vectors have been identified using molecular-based tools. Genomic diversity of phytoplasma groups appears to be correlated with their sharing common insect vectors, host plants, or both in nature. The level of exchange of genetic information among phytoplasma strains in a given group is determined by three-way, vector-phytoplasma-plant interactions. A putative mechanism for the creation of new ecological niches and the evolution of new ecospecies is proposed.     

hpRNA的茎环比例对RNA介导的病毒抗性产生的影响

 以马铃薯Y病毒坏死株系(PVY^N)的外壳蛋白(coat protein,CP)基因3'端50bp片段为hpRNA的茎,以pUC19不同长度的序列为hpRNA的环,构建了茎环比例分别为4:1、2:1、1:1、1:2、1:4和1:8的植物表达载体。利用农杆菌介导法转化烟草品种NC89,获得了多种转基因植株。室内抗病性检测发现:不同茎环比例的hpRNA介导的病毒抗性效率不同;茎环比例为4:1、2:1和1:1时效率较高,抗性植株的比例达60%左右;随着环长度的逐渐增加,抗性植株的比例逐渐降低;当茎环比例为1:8时,抗性植株的比例仅为9.52%。Southern blot分析结果表明:外源基因已整合于烟草的基因组中,且转基因植株的抗病性与转基因的拷贝数之间无明显的相关性。Northern blot分析结果表明:目的片段转录产物的积累量与植株的抗病性呈负相关,证明所获得的抗病性是RNA介导的。     

水分胁迫下小麦TaWSI18基因的电子克隆及生物信息学分析

利用电子克隆和RT-PCR技术,从干旱胁迫的小麦叶片中分离出4条新的具有高度同源性的WSI18基因,即TaWSI18-1、TaWSI18-2、TaWSI18-3和TaWSI18-4(Gene Bank登陆号分别为:KP226849、KP226850、KP226851和KP226852);其开放阅读框均为678 bp,编码225个氨基酸。生物信息学分析表明,该类蛋白具有亲水性,存在多个磷酸化位点,无信号肽结构域及剪切位点,定位于细胞质中,无跨膜结构域;二级结构分析表明,该蛋白α螺旋和无规则卷曲的比例达到90%以上;同源性比较及聚类分析表明,小麦TaWSI18蛋白同无芒雀麦(Bromus inermis,BAN15016)WSI18蛋白和二穗短柄草(Brachypodium distachyon,XP 003569641)LEA3蛋白具有高度的同源性,相似性分别为94%和70%。小麦TaWSI18基因编码蛋白的氨基酸序列的N端存在与LEA蛋白的N端区域高度保守的序列。表明小麦TaWSI18蛋白的性质与LEA3蛋白的相似,说明了小麦的TaWSI18蛋白可能与LEA3蛋白存在相同或相似的功能,为进一步研究其在小麦水分胁迫下的功能奠定基础。     

葡萄座腔菌(Botryosphaeria dothidea)全基因组分泌蛋白的预测及功能分析

 葡萄座腔菌(Botryosphaeria dothidea)是引起多种果树和林木枝干溃疡病和果实腐烂病的重要病原菌。为明确葡萄座腔菌的致病机理,采用SignalP、WoLF PSORT、TMHMM、GPI-SOM等软件,对葡萄座腔菌全基因组14 998条蛋白序列进行了分泌蛋白预测和功能分析。结果表明B.dothidea全基因组编码蛋白中有851条序列具有典型的分泌蛋白特征,占蛋白总数的5.67%,其长度主要分布在100~700个氨基酸范围内。信号肽的分析结果表明,以19个氨基酸为信号肽的分泌蛋白数目最多,非极性氨基酸丙氨酸(A)在信号肽中的使用频率最高,而有带电侧链的天冬氨酸(D)和谷氨酸(E)使用频率最低,信号肽-3和-1位置的氨基酸相对保守,其切割位点属于典型的A-X-A型。对分泌蛋白功能预测结果表明,578个分泌蛋白获得了功能注释,其功能主要涉及碳水化合物的运输、代谢过程,蛋白翻译后修饰和氨基酸代谢、运输过程。分泌蛋白中效应蛋白(effector)预测结果表明,B. dothidea分泌蛋白中共有119个潜在的效应蛋白,其中11个可被PHI数据库注释到,其与引起其他植物病原菌致病力变化的致病效应蛋白具有较高的相似性。碳水化合物活性酶类(CAZymes)的预测结果表明,B. dothidea分泌蛋白组中共有279个CAZymes,其中GHs家族最多。这些结果的获得为今后进一步筛选B. dothidea的效应蛋白,明确B. dothidea的致病机理,以及筛选寄主新的抗性基因提供了必要的基础。     

Positional effect of gene insertion on genetic stability of a clover yellow vein virus-based expression vector

The stability of the inserted genes in the viral expression vector varied depending on the sequence introduced and the position of insertion. Infectious cDNA to Clover yellow vein virus (pClYVV) was modified to insert a foreign gene at two independent sites: one, along with a polylinker, between the NIb and CP genes (pClYVV/CP/W) and the other between P1 and HC-Pro (pClYVV-Pst/CP). The green fluorescent protein (GFP) gene was inserted into either pClYVV/CP/W or pClYVV-Pst/CP. GFP gene was stably maintained and expressed in both vectors following serial passages in plants. Progeny viruses from both constructs accumulated in similar amounts and at rates of 70%–80% of that of the wild-type virus. On the other hand, progeny viruses carrying the human interferon- (hIFN) gene cloned in pClYVV-Pst/CP were genetically unstable owing to frequent deletions of the cloned gene during passage through plants. In contrast, the hIFN sequence cloned in pClYVV/CP/W was stably maintained in viruses after several passages in broad bean plants, and the progeny virus accumulated at the rate of about 50%–100% of that of the wild-type virus. The nucleotide sequence analyses indicated that the genetic instability of the inserted sequence results from homologous recombination of viral vector and inserted DNA sequences; it is not due to the inserted sequence alone.     

应用基因枪法获得抗大麦黄矮病毒转基因小麦

 以我国特有的大麦黄矮病毒GPV株系的外壳蛋白(CP)基因为材料,设计合成了分别含有Act启动子或Emu启动子的植物表达载体pPPI2、pPPI3和pPPI5。采用基因枪法分别转化小麦幼胚和愈伤组织。诱导成苗后进行PCR检测,T₀代阳性率为18%。对转基因苗的后代进行进一步检测,部分转基因苗阳性株至T₃代PCR检测阳性率为100%,PCR结果CP探针杂交呈阳性反应,序列测定结果与GPV CP基因序列一致,表明GPV CP基因已整合到小麦基因组中。室内抗病性鉴定结果,虽然转基因植株全部发病,但对大麦黄矮病毒GPV株系具有一定的延迟发病作用。     

Classical and molecular genetics of <Emphasis Type="Italic">Bremia lactucae</Emphasis>, cause of lettuce downy mildew

Lettuce downy mildew caused by Bremia lactucae has long been a model for understanding biotrophic oomycete–plant interactions. Initial research involved physiological and cytological studies that have been reviewed earlier. This review provides an overview of the genetic and molecular analyses that have occurred in the past 25 years as well as perspectives on future directions. The interaction between B. lactucae and lettuce (Lactuca sativa) is determined by an extensively characterized gene-for-gene relationship. Resistance genes have been cloned from L. sativa that encode proteins similar to resistance proteins isolated from other plant species. Avirulence genes have yet to be cloned from B. lactucae, although candidate sequences have been identified on the basis of motifs present in secreted avirulence proteins characterized from other oomycetes. Bremia lactucae has a minimum of 7 or 8 chromosome pairs ranging in size from 3 to at least 8 Mb and a set of linear polymorphic molecules that range in size between 0.3 and 1.6 Mb and are inherited in a non-Mendelian manner. Several methods indicated the genome size of B. lactucae to be ca. 50 Mb, although this is probably an underestimate, comprising approximately equal fractions of highly repeated sequences, intermediate repeats, and low-copy sequences. The genome of B. lactucae still awaits sequencing. To date, several EST libraries have been sequenced to provide an incomplete view of the gene space. Bremia lactucae has yet to be transformed, but regulatory sequences from it form components of transformation vectors used for other oomycetes. Molecular technology has now advanced to the point where rapid progress is likely in determining the molecular basis of specificity, mating type, and fungicide insensitivity.     

Development of PPO inhibitor-resistant cultures and crops

Recent progress in the development of protoporphyrinogen oxidase (PPO, Protox) inhibitor-resistant plant cell cultures and crops is reviewed, with emphasis on the molecular and cellular aspects of this topic. PPO herbicide-resistant maize plants have been reported, along with the isolation of plant PPO genes and the isolation of herbicide-resistant mutants. At the same time, PPO inhibitor-resistant rice plants have been developed by expression of the Bacillus subtilis PPO gene via targeting the gene into either chloroplast or cytoplasm. Other attempts to develop PPO herbicide-resistant plants include conventional tissue culture methods, expression of modified co-factors of the protoporphyrin IX binding subunit proteins, over-expression of wild-type plant PPO gene, and engineering of P-450 monooxygenases to degrade the PPO inhibitor.     

Some coat protein constituents from strawberry latent ringspot virus expressed in transgenic tobacco protect plants against systematic invasion following root inoculation by nematode vectors

The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression inAgrobacterium tumefaciens-transformedNicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG initiation codon sequences added at the 5-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5 S/G site and a TAG stop codon sequence added at the 3-proximal S/G site. The P, L and S proteins were expressedin planta to a maximum concentration of 0.01 % of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S1 and S2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.     

Baculoviruses as vectors for foreign gene expression in insect cells

Recombinant DNA technology has enabled the expression of a large number of eukaryotic genes in vitro. There has been growing interest in recent years in the development of baculovirus expression vectors as an easily manipulated gene expression system. Both virus and insect cell culture are relatively easy to handle, and biologically active proteins have been produced abundantly from a variety of eukaryotic, prokaryotic and viral genes. This review describes the molecular biology of baculoviruses and some of the current applications of the baculovirus expression system in insect cells. Specific emphasis is placed on those features relevant to the use of this system in pesticide research.     

The diverse roles of NB-LRR proteins in plants

Plant innate immunity relies on specialised immune receptors that can detect and defend against a wide variety of microbes. The first group of receptors comprises the transmembrane pathogen- or pattern-recognition receptors (PRRs), which respond to slowly evolving pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs). The second group of immune receptors is formed by the polymorphic disease resistance (R) proteins that detect microbe-derived effector proteins. Most R proteins are members of the nucleotide binding leucine-rich repeat (NB-LRR) class. Although this class comprises one of the biggest protein families in plants, relatively few have been functionally characterised to date. The question rises whether all NB-LRRs function as immune receptors, or that they might have alternative functions. The answer is: yes, they do have alternative functions that are different from the immune receptor function. This review summarises the current knowledge about non-immune receptor signal transduction functions of NB-LRRs in plants.     

What proteomic analysis of the apoplast tells us about plant–pathogen interactions

Plant pathogens have developed different strategies during their evolution to infect and colonize their hosts. In the same way, plants have evolved different mechanisms acting against potential pathogens trying to infect and colonize their tissues. Regulation of a wide variety of proteins is required in order to perceive the pathogen and to activate the plant defence mechanisms. The apoplast is the first compartment where these recognition phenomena occur in most plant–pathogen interactions, allowing the exchange of different molecules and facilitating inter‐ and intracellular communication in plant cells. Proteomic analysis of the apoplast in recent years has found the initial biochemical responses involved in pathogen recognition and early defence responses. However, this proteomic approach requires some specific experimental conditions to obtain an extract free of cytoplasmic proteins and nonprotein contaminants that affect the subsequent stages of separation and quantification. Obtaining the highest proportion of proteins from the apoplastic space in infected tissues requires different steps such as extraction of apoplastic washing fluids and preparation of total secreted proteins (protein precipitation, solubilization, separation and digestion). Protein identification using mass spectrometry techniques and bioinformatics tools identifying peptides for the extracellular exportation is required to confirm the apoplastic location. This review compiles the most commonly used techniques for proteomic studies, focusing on the early biochemical changes occurring in the apoplast of plants infected by a wide range of pathogens. The scope of this approach to discover the molecular mechanisms involved in the plant–pathogen interaction is discussed.     

Molecular cloning and characterization of disease-resistance gene candidates of the nucleotide binding site (NBS) type from Cocos nucifera L

In this study, degenerate primers were used to amplify nucleotide-binding site (NBS)-type sequences from coconut ecotypes either resistant or susceptible to lethal yellowing. Genomic DNA fragments of approximately 500–700 bp were obtained and sequenced. Phylogenetic analysis of these fragments revealed that they clustered in seven different clades. We also found that all CnRGC sequences were grouped within the non-TIR-NBS-LRR subclass of NBS-LRR genes. The expression analysis revealed changes in expression profiles in response to salicylic acid (SA) and a constitutive expression profile in plants untreated with SA. This is the first large scale analysis of NBS-type sequences in coconut palm.     

Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.     

转录后基因沉默

 转录后基因沉默(PTGS)普遍存在于生物界,如植物的共抑制、源于病原的RNA介导的病毒抗性、真菌的基因沉默和动物的RNA干扰等。这类现象有许多共同特点,如都是以向细胞内引入核酸(转基因、双链RNA或病毒RNA)为诱因,依赖RNA的RNA聚合酶(RdRP)活性与基因沉默密切相关,在发生基因沉默的细胞中大多存在特定长度的小分子RNA (21~25 nt),PTGS导致细胞质内mRNA的特异性降解,不同生物的PTGS相关基因及产物具有很高的相似性,基因沉默能够在细胞间传播并能以表型遗传的方式传递给下一代等。这说明各种生物的转录后基因沉默可能有相似的遗传起源,是一种抵抗外来核酸(如病毒和转座子)入侵的共同的防御机制     

蚜虫传播非持久性病毒的取食行为调控机制

蚜虫能够传播上百种植物病毒，是最重要的农业介体昆虫之一。蚜虫在刺探和取食植物过程中，唾液组分会连同附着在口针中的病毒粒子一同被分泌进入植物内，在调节植物诱导抗性、病毒侵染扩散、介体昆虫行为等过程中均有重要作用。本文围绕蚜虫传播病毒和获取病毒2个关键过程，总结分析了蚜虫独特的刺吸取食行为与传毒效率和获毒效率之间的联系；针对取食活动中关键的唾液蛋白在调控植物免疫抗性、帮助病毒侵染过程中的功能，阐述了蚜虫高效传播非持久病毒的分子基础；针对蚜虫的获毒过程，综述了病毒侵染植物间接调控蚜虫趋向和行为的作用方式。这些研究的开展将为解释蚜虫和病毒协同侵染的分子机制以及有效开展基于蚜虫取食行为调控的病虫害防控新技术提供思路。     

Genomic variability and plasticity of Pseudomonas causing coffee leaf spots in Minas Gerais state,Brazil

Foliar spots caused by Pseudomonas coronafaciens pv. garcae (Pcg), Pseudomonas amygdali pv. tabaci (Pat) and Pseudomonas cichorii (Pch) are major bacterial diseases that can reduce coffee production. However, little is known about the genetic diversity and molecular mechanisms underlying the pathogenicity to coffee plants of these bacteria. In this study, genome sequences of Pcg, Pat and Pch strains isolated from coffee plants in Minas Gerais state, Brazil, were used to assess their variability and plasticity, and compare their type III secretion system (T3SS) and apoplastic effector repertoires as well as tabtoxin biosynthetic/detoxification genes. Genomic diversity was found for all three phytopathogens, among which Pch possesses the highest number of exclusive proteins. The Pcg genome is the most stable whereas that of Pch is the most plastic, which is related to their host ranges. When compared with those of Pseudomonas syringae pv. tomato DC3000, hrp/hrc gene sequences are more conserved in Pcg and Pat than in Pch, which also possesses the smallest T3SS and the largest apoplastic effector repertoires. The only T3SS effector family common to all three pathogens is AvrE, suggesting that, as for other plant–Pseudomonas interactions, it may play a crucial role for pathogenicity towards coffee plants. Apoplastic proteins associated with maintaining the redox balance and degrading proteins/peptides not previously described as important in plant–bacteria interactions were found. Gain/loss of the tabtoxin biosynthetic cluster with retention of the antitoxin gene was observed, indicating that tabtoxin production is not a limiting factor for the occurrence of mixed infections.