Timing and extent of hypersensitive response are critical to restrict local and systemic spread of Pepper mild mottle virus in pepper containing the L 3 gene

Amino acid changes in Pepper mild mottle virus (PMMoV) coat protein (CP) that enhance, decrease, or nullify the resistance-inducing activity in Capsicum plants carrying the L ³ gene have been identified. In this study, molecular events underlying the L ³ -gene-mediated resistance were analyzed through the expression of hypersensitive response (HR)-related genes, HSR203J-Cc and HIN1-Cc, and defense-related genes, PR1-Cc and PR4b-Cc, upon infection with PMMoV CP mutants. The expression kinetics of the genes correlated with the degree of restriction of virus distribution in the inoculated leaves. The results suggest that the timing and extent of HR are critical factors to restrict virus spread both locally and systemically in L ³ -gene-mediated resistance.The nucleotide sequence data reported are available in the DDBJ/GenBank/EMBL databases under accession numbers AB162220 (HSR203J-Cc), AB162221 (HIN1-Cc), AB162222 (PR1-Cc), and AB162223 (PR4b-Cc)     

Pathogenesis-related gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae

Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between Pathogenesis Related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, and OsPR1c genes were cloned and their induction was analyzed, in addition to the OsPR10a gene, at the juvenile and adult stages in response to a wildtype Xoo strain that induces a resistance response (incompatible interaction) and an isogenic mutant Xoo strain that does not (compatible interaction). We found that the adult stage leaves are more competent to express these OsPR1 genes and that the Xa21 locus is required for the highest levels of induction.     

Histochemical studies on the accumulation of reactive oxygen species (O2 and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression

Large-scale cDNA-AFLP profiling identified numerous genes with increased expression during the resistance response of wheat to the Septoria tritici blotch fungus, Mycosphaerella graminicola. To test whether these genes were associated with resistance responses, primers were designed for the 14 that were most strongly up-regulated, and their levels of expression were measured at 12 time points from 0 to 27 days after inoculation (DAI) in two resistant and two susceptible cultivars of wheat by real-time quantitative polymerase chain reaction. None of these genes was expressed constitutively in the resistant wheat cultivars. Instead, infection of wheat by M. graminicola induced changes in expression of each gene in both resistant and susceptible cultivars over time. The four genes chitinase, phenylalanine ammonia lyase, pathogenesis-related protein PR-1, and peroxidase were induced from about 10- to 60-fold at early stages (3 h–1 DAI) during the incompatible interactions but were not expressed at later time points. Nine other genes (ATPase, brassinosteroid-6-oxidase, peptidylprolyl isomerase, peroxidase 2, 40S ribosomal protein, ADP-glucose pyrophosphorylase, putative protease inhibitor, methionine sulfoxide reductase, and an RNase S-like protein precursor) had bimodal patterns with both early (1–3 DAI) and late (12–24 DAI) peaks of expression in at least one of the resistant cultivars, but low if any induction in the two susceptible cultivars. The remaining gene (a serine carboxypeptidase) had a trimodal pattern of expression in the resistant cultivar Tadinia. These results indicate that the resistance response of wheat to M. graminicola is not completed during the first 24 h after contact with the pathogen, as thought previously, but instead can extend into the period from 18 to 24 DAI when fungal growth increases dramatically in compatible interactions. Many of these genes have a possible function in signal transduction or possibly as regulatory elements. Expression of the PR-1 gene at 12 h after inoculation was much higher in resistant compared to susceptible recombinant-inbred lines (RILs) segregating for the Stb4 and Stb8 genes for resistance. Therefore, analysis of gene expression could provide a faster method for separating resistant from susceptible lines in research programs. Significant differential expression patterns of the defense-related genes between the resistant and susceptible wheat cultivars and RILs after inoculation with M. graminicola suggest that these genes may play a major role in the resistance mechanisms of wheat.     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

Mycorrhiza induced resistance in potato plantlets challenged by Phytophthora infestans

Biological control of soil-borne pathogens by arbuscular mycorrhizal (AM) fungi has been repeatedly demonstrated. However, their role in the control of above-ground hemibiotrophic pathogens is less conclusive. Here, we investigated in vitro the impact of an AM fungus on Phytophthora infestans in potato plants. The leaf infection index was decreased in mycorrhizal potato plants. Real-Time Quantitative PCR revealed the induction of two pathogenesis related genes (PR1 and PR2) in the leaves of mycorrhizal plants shortly after infection with P. infestans. These results suggested a systemic resistance in mycorrhizal plants, related to the priming of the two PR genes in potato.     

MPK4甲基化调控水杨酸信号介导的拟南芥对齐整小核菌菌株SC64的抗性机制

为提升齐整小核菌Sclerotium rolfsii菌株SC64作为生物除草剂的应用潜力，解析植物对其的防御机制，通过比较6种生态型拟南芥Arabidopsis thaliana(Col-0、Ms-0、Gr-1、Se-0、Tu-0和Wa-1)被菌株SC64侵染后病理特征、茎秆木质素水平以及抗病相关基因表达的差异，筛选出抗性最高和抗性最弱的典型生态型拟南芥，通过施加0.1 mmol/L外源水杨酸来验证其在拟南芥防御菌株SC64侵染过程中对木质素代谢及病理的调节作用，并比较分析6种生态型拟南芥水杨酸上游调控通路基因MPK4启动子区域的甲基化差异。结果显示，6种生态拟南芥中Col-0的木质素化程度最高，被菌株SC64侵染时木质素代谢通路基因CAD4和PAL3表达上调最显著，与其抗病能力最强相吻合，而Ms-0的病理表型和抗病能力最弱。外源水杨酸预处理使具典型抗性的生态型拟南芥Col-0受菌株SC64侵染后发病程度加重，木质素化程度减弱，同时木质素代谢通路基因MYB46、LAC17、PAL3和CAD4被菌株SC64侵染后的表达上调程度均显著减弱。另外，6种生态型拟南芥MPK4基因启动子区域CHH...     

Ca2+对Cd2+胁迫下板蓝根种子萌发及幼苗抗氧化酶活性的影响

研究了Cd²⁺（10 mg·L^-1、30 mg·L^-1）胁迫下不同浓度Ca²⁺（0、80、160、320 mg·L^-1）对板蓝根种子萌发、幼苗抗氧化酶系统及蛋白质含量的影响。结果表明：低浓度Ca²⁺（80、160 mg·L^-1）可缓解Cd²⁺毒害,显著提高板蓝根种子的发芽率、发芽势、发芽指数、活力指数,促进蛋白质含量的增加,提高SOD、POD、CAT活性,且160　mg·L^-1 Ca²⁺缓解效果最好,缓解能力随Cd²⁺浓度的升高有所下降;高浓度Ca²⁺（320 mg·L^-1）与Cd²⁺作用,反而抑制了板蓝根种子的萌发,幼苗的POD、SOD、CAT活性及蛋白质含量下降。低浓度Ca²⁺可以显著提高板蓝根的抗性,对Cd²⁺毒害起缓解作用,高浓度的Ca²⁺与Cd²⁺对板蓝根种子起协同毒害作用。     

棉铃虫YTHDF1基因的表达模式及在核型多角体病毒侵染中的作用

为明确N6-甲基腺苷（N6-methyladenosine,m⁶A）修饰在棉铃虫核型多角体病毒（Helicoverpa armigera nucleopolyhedrovirus,HearNPV）侵染棉铃虫中的作用,根据基因组和转录组数据,对棉铃虫m⁶A结合蛋白基因YTHDF1（YTH domain-containing family protein 1）进行鉴定和分析,利用实时荧光定量PCR技术检测棉铃虫YTHDF1基因的时空表达模式、HearNPV处理后的表达变化情况以及RNA干扰效率,并调查该基因下调后对HearNPV复制及感染HearNPV棉铃虫死亡情况的影响。结果显示,棉铃虫YTHDF1基因开放阅读框全长为2 019 bp,编码672个氨基酸,含有1个保守的YTH结构域,其氨基酸序列与斜纹夜蛾Spodoptera litura同源物序列一致性达87.52%。YTHDF1基因在棉铃虫不同发育阶段均有表达,其中在进入5龄期24 h幼虫中的表达量最高,在2龄取食期幼虫中的表达量最低。YTHDF1基因的空间表达谱呈现一定的组织特异性,在幼虫血细胞和成虫头部的表达量最高。HearNPV侵染棉铃虫后YTHDF1基因上调表达,经RNA干扰使该基因下调后显著抑制了HearNPV多角体蛋白基因polyhedrin的表达并延迟了感染HearNPV棉铃虫的死亡时间。表明YTHDF1基因在HearNPV侵染棉铃虫的过程中发挥着重要作用。     

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance (R) genes. In Arabidopsis, some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR-1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR1 and EDS1.     

Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis

Fusarium graminearum, Fusarium culmorum and Fusarium cerealis are major causal agents of Fusarium Head Blight (scab) which is a disease of global significance in all cereal growing areas. These fungi produce trichothecene mycotoxins, principally nivalenol (NIV) and deoxynivalenol (DON). Genes Tri13 and Tri7 from the trichothecene biosynthetic gene cluster convert DON to NIV (Tri13) and NIV to 4-acetyl-NIV (Tri7). We have developed positive–negative PCR assays based on these two genes, which accurately indicate a DON or NIV chemotype in F. graminearum, F. culmorum and F. cerealis. These assays are useful in assessing the risk of trichothecene contamination, and can be informative in epidemiological studies. All NIV chemotype isolates studied have functional copies of both Tri13 and Tri7, and all DON-producing isolates have both genes disrupted or deleted. We have identified several mutations in these genes, which are conserved across F. graminearum lineage, RAPD and SCAR groupings and between the three species. There appears to be evidence of inter-species hybridisation within the trichothecene biosynthetic gene cluster.     

Rwt4 , a wheat gene for resistance to Avena isolates of Magnaporthe oryzae , functions as a gene for resistance to Panicum isolates in Japan

Rwt4 (synonym of Rmg1), a temperature-insensitive gene for resistance to Avena isolates of Magnaporthe oryzae, was identified in wheat cultivar Norin 4 in a seedling assay. The significance of Rwt4 was evaluated using flag leaves of wheat cultivars. At high temperature, Norin 4 was completely resistant to Avena isolate Br58, while Chinese Spring, a noncarrier of Rwt4, was susceptible. Genetic analysis of F₂ plants derived from Norin 4 × Chinese Spring indicated that the resistance of flag leaves of Norin 4 to the Avena isolate is conditioned by a single major gene. Segregation analysis of F₃ seedlings derived from the F₂ plants showed that the major gene is actually Rwt4. These results suggest that Rwt4 is effective against Avena isolates throughout the growth stages. Furthermore, screening of Pyricularia isolates from various hosts suggested that Panicum isolates are possible carriers of the corresponding avirulence gene, PWT4. Segregation analyses of F₂ and F₃ seedlings showed that Panicum isolates actually carry PWT4, and, therefore, that Rwt4 is also effective against Panicum isolates. On the other hand, none of the Oryza, Setaria, Triticum, and Lolium isolates tested was a carrier of PWT4. The significance of this finding is discussed from the viewpoint of epidemics of blast disease on wheat.     

The influence of carotenoid biosynthesis modification on the Fusarium culmorum and Fusarium oxysporum resistance in flax

Flax engineering to yield increased resistance to pathogens is the goal of this study. Since carotenoids act as antioxidants it is thus postulated that the accumulation of a higher quantity of these compounds in the transgenic plants might improve their resistance to pathogen infection.Our approach was based on the generation of transgenic flax overproducing carotene and analysis of its susceptibility to Fusarium infection. For transformation bacterial gene – crtB was used. As expected, transgenic plants showed increased resistance against pathogen infection.The impact of carotenoids on plant resistance to infection was verified by generation and analysis of transgenic flax with decreased content of carotene. The transgenic plants were obtained by suppression of endogenous flax gene coding for lycopene β-cyclase. Plant analysis revealed decrease in carotene content, however, an unexpected increase in resistance against Fusarium infection was detected. Further analysis of metabolites in the plants revealed that an increase in accumulation of other terpenoids and tocopherols, squalene and menthol were among them. Thus, it is suggested that repression of carotene synthesis results in the redirecting of substrates to other branches of isoprenoids synthesis.We conclude that a general level of antioxidants rather than the presence of any particular compound is the most important factor in resistance of the flax plant to pathogen infection.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

烟蚜热激蛋白Hsp90基因的克隆及在UV-B胁迫下的表达分析

为探讨UV-B胁迫对烟蚜Myzus persicae热激蛋白Hsp90基因表达量的影响,采用RT-PCR与RACE技术克隆了烟蚜热激蛋白Hsp90基因的全长,并对其进行生物信息学分析,利用实时荧光定量PCR技术研究了烟蚜Hsp90基因在不同时长UV-B胁迫下的表达量变化。结果表明,烟蚜Hsp90基因的cDNA全长为2 670 bp,编码728个氨基酸,编码蛋白质的相对分子量为82.6 kD,等电点为4.95,获得的氨基酸序列具有Hsp90蛋白家族的1个签名序列及C末端MEEVD基序,推测其属于胞质型热激蛋白。系统进化树结果显示,烟蚜Hsp90与其它昆虫Hsp90具有很高的相似性。实时荧光定量PCR结果表明,不同时长UV-B胁迫下烟蚜Hsp90均有表达,随着照射时间延长,Hsp90表达量表现为先上升后下降的趋势;与对照相比,照射时间为15、30、60、90和120 min时,Hsp90表达量均显著升高,且在60 min时Hsp90表达量达最大,是对照组的2.05倍。表明Hsp90基因在不同时长UV-B胁迫下差异表达,在烟蚜适应紫外胁迫的分子机制中具有重要作用。