Differential expression of molecular rust resistance components have distinctive profiles in <Emphasis Type="Italic">Coffea arabica</Emphasis> - <Emphasis Type="Italic">Hemileia vastatrix</Emphasis> interactions

Countering the economic hurdle caused by coffee leaf rust disease is most appealing at this time as it has posed a major threat to coffee production around the world. Establishing differential expression profiles at different times following pathogen invasion in both innate and acquired immunities unlocks the molecular components of resistance and susceptibility. Suppression subtractive hybridization (SSH) was used to identify genes differentially over-expressed and repressed during incompatible and compatible interactions between Coffea arabica and Hemileia vastatrix. From 433 clones of expressed sequence tags (ESTs) sequenced, 352 were annotated and categorized of which the proportion of genes expressed during compatible interaction were relatively smaller. The result showed upregulation and downregulation of various genes at 12 and 24 h after pathogen inoculation in both interactions. The use of four different databases in searching for gene homology resulted in different number of similar sequences. BLASTx against EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute) database being with the maximum (100%) hits for all the annotated sequences. RT-qPCR analysis of seven resistance-signaling genes showed similar expression patterns for most of the genes in both interactions, indicating these genes are involved in basal (non-specific) defense during which immune reactions are similar. Using SSH, we identified different types of resistance related genes that could be used for further studies towards resistant cultivar development. The potential role of some of the resistance related proteins found were also discussed.     

野生甜瓜‘云甜-930’与白粉病菌互作的基因表达特征

 利用cDNA-AFLP技术, 对甜瓜抗白粉病品种‘云甜-930’在接种Podosphaera xanthii生理小种2F.后的基因表达谱进行分析。256对引物共产生188个具良好多态性的转录本(TDF), 其中109个上调表达, 79个下调表达。经过对差异片段的回收、克隆、测序分析, 最终得到60个EST。Blastx比对和功能分类分析表明, 参与物质合成与代谢的属第一大类, 占48%, 其他主要涉及物质运输(12%)、防御系统(12%)、转录调控(8%)、能量代谢(8%)、信号转导(4%)等, 7条EST(8％) 与未知功能蛋白同源性较高。选取与代谢、抗病防御、信号转导及蛋白转运等相关的4个差异基因TDF12(SEH)、TDF67(SAMDC)、TDF76(CDPK)和TDF82(PDR8)进行qRT-PCR验证, 结果显示其表达模式符合cDNA-AFLP表达谱, 同时表明这些基因可能参与了甜瓜与白粉病菌的互作过程。     

Molecular Diversity of Agriculturally Important Aspergillus Species

Although Aspergillus species are not usually considered as serious plant pathogens, Aspergilli are frequently encountered in plant products. The most important consequence of their presence is mycotoxin contamination. The main mycotoxins produced by Aspergilli are the aflatoxins, ochratoxin A and patulin, which are produced by a variety of Aspergillus species in different plant commodities. Phylogenetic analysis of sequences of the ribosomal RNA gene cluster is useful for clarifying taxonomic relationships among toxigenic Aspergilli causing pre- and postharvest contamination of agricultural products. Molecular data has enabled us to clarify the taxonomy of black Aspergilli, A. flavus and its relatives, and sections Circumdati and Clavati, which include ochratoxin and patulin-producing species. Phylogenetically unrelated species were found to produce the same mycotoxins, indicating that mycotoxin-producing abilities of the isolates have been lost (or gained) several times during the evolution of the genus. The data also indicate that biosynthetic gene-based probes are necessary for molecular detection of these mycotoxin-producing organisms. The organisation of the biosynthetic genes of patulin and ochratoxins is unknown, although experiments are in progress in several laboratories to clarify the genetic background of biosynthesis of these mycotoxins. Identification of biosynthetic genes responsible for mycotoxin production is essential for clarifying the evolution of mycotoxin biosynthesis in Aspergilli, and to develop specific gene probes for the detection of mycotoxin-producing Aspergilli in agricultural products.     

链孢粘帚霉寄生核盘菌菌核差异表达基因的筛选及分析

为克隆和研究链孢粘帚霉Gliocladium catenulatum寄生核盘菌菌核的相关基因,应用抑制消减杂交技术构建了cDNA消减文库并进行了筛选。通过PCR技术从文库中共筛选到1315个阳性克隆,克隆中插入片段大小主要集中于300～600bp之间。随机挑取120个克隆,经测序和同源性分析,获得60条有效序列,其中部分序列所编码的血红素加氧酶、核糖体蛋白L11、细胞色素P450及热激蛋白等均参与机体对胁迫条件的应答反应。11条序列在NCBI数据库中未找到显著匹配的序列,可能为新基因片段。分别将寄生于核盘菌菌核上的粘帚霉cDNA和粘帚霉与核盘菌纯培养的cDNA混合物经RasⅠ酶切后进行标记作为探针,利用反向Northern杂交技术验证了所选取的25条序列全部为差异表达基因片段。     

大豆根组织受尖孢镰刀菌侵染后基因表达反应的cDNA-AFLP分析

 为了阐明大豆受尖孢镰刀菌(Fusarium oxysporum,Fo)侵染后在基因转录水平上的感病反应,本研究用cDNA-AFLP方法,对Fo浸根接种后大豆根组织进行了基因转录谱的银染法检测分析。结果表明,在测定的大约1 000个大豆cDNA扩增片段(TDFs)中,16个是差异性表达的,其中9个受Fo诱导上调表达,7个被抑制下调表达。对这些差异性表达的TDFs进行克隆、测序及其同源性进行分析,发现其中6个具有已知或推断的生物学功能,包括钙调素结合蛋白(CaMBP)、BURP结构域蛋白和羟基多花碱-羟基烷宁转移酶等,其余10个功能未知。这些可能参与大豆感病性的差异性表达新基因片段的发现,为开展大豆感病性功能基因组学研究提供了材料。     

Screening for Differential Gene Expression in Atropa belladonna Leafy Gall Induced Following Rhodococcus fascians Infection

To better characterise at the molecular level the nature of plant responses to infection by Rhodococcus fascians PCR-based differential display patterns of Atropa belladonna leafy gall (LG) and non-infected plant tissues were compared. Six differentially expressed genes were identified and their altered expression was confirmed by RT-PCR. Three of them corresponded to up-regulated genes which encode proteins involved in plant defence. The three remaining cDNA fragments which correspond to down-regulated genes in LG, encoded proteins with similarity to a multicystatin, a miraculin and a methallothionein-like protein, respectively. Upon elimination of the bacteria from infected plant tissue, the expression of up-regulated genes was maintained, whereas expression of down-regulated genes resumed suggesting a potential role of these up-regulated genes in plant growth and development.     

DNA methylation changes in fusarium wilt resistant and sensitive chickpea genotypes (Cicer arietinum L.)

DNA methylation plays an important role in the regulation of gene expression in biotic and abiotic stresses. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in seven resistant and sensitive chickpea genotypes following inoculation with Fusarium oxysporum f. sp. ciceris. In all, 27468 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using nine selective primer pairs. DNA methylation was evaluated in leaves, stems and roots in control and inoculated plants. Extensive cytosine methylation alterations were found in the pathogen-treated genotypes compared with the corresponding control, including hypermethylation and demethylation as well as the potential conversion of methylation types. For all genotypes, the percentage of demethylated sites were more than methylated sites in infected plants compared with the corresponding control. No significant differences were observed for banding patterns in infected and control leaf tissues, while the differences between percentage of unchanged, methylated and demethylated sites were significant in stem and root tissues. The total numbers of methylated polymorphic bands ranged from 137 to 154 bands in Sel95th1716 and Arman, accounting for 36.81%–44.64% of all bands, respectively. Ten fragments that were differentially amplified between infected and control plants were isolated and sequenced in three tissues separately. Most of sequenced fragments showed homology with disease related genes in GenBank. The results suggest that significant differences in cytosine methylation exist between resistant and sensitive chickpea genotypes, and that hypermethylation or hypomethylation of specific genes may be involved in the chickpea resistance to Fusarium wilt.     

Insights on gene expression response of a characterized resistant genotype of Solanum commersonii Dun. against Ralstonia solanacearum

Solanum commersonii is a wild species related to the cultivated potato. Some S. commersonii genotypes have been proven to be resistant to the pathogenic bacteria Ralstonia solanacearum, which causes damage in potato and other economically important crops. Here an expression analysis of the response of a resistant S. commersonii genotype against R. solanacearum was performed using microarrays. The aims of this work were to elucidate the molecular processes involved in the interaction, establish the timing of the response, and contribute to identify genes related to the resistance. The response to the treatment was already initiated at 6 h post-inoculation (hpi) and was established at 24 hpi; during this period, a high number of genes was differentially expressed and several candidate genes for the resistance of S. commersonii to R. solanacearum were identified. At an early stage, the photosynthetic process was highly repressed and several genes encoding proteins related to reactive oxygen species (ROS) production were differentially expressed. The induction of ERF and ACC-oxidase genes related to the ethylene pathway and PR1 related to the salicylic acid pathway suggested the induction of both pathways, and back up the previously reported hemibiotrophic nature of the pathogen. Five genes related to plant defence and observed to be differentially expressed at the first two time points were validated by real time PCR. This work gives a glimpse to the molecular processes involved in S. commersonii resistance and identifies the species as a valuable genetic source for potato breeding against bacterial wilt.     

Identification of genes associated with phytoplasma resistance through suppressive subtraction hybridization in Chinese jujube

Jujube witches' broom (JWB) is a destructive disease for Chinese jujube caused by phytoplasma. A suppression subtractive hybridization library of resistant cultivar ‘Xingguang’ was constructed under phytoplasma stress to identify genes related to JWB resistance. 77 of 200 unique expressed sequence tags had significant sequence homologies and were classified into 10 functional groups. The most abundant group was disease/defense (20.8%), which was consistent with the phytoplasma stress. These differentially expressed genes provide the groundwork for addressing the plant–phytoplasma interaction. Meanwhile, the expression of five selected genes (TLP, PR10, HSP70, ERF, kinase-related protein) was confirmed to upregulate at different infection periods.