Host‐induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust

Rust fungi are devastating plant pathogens and cause a large economic impact on wheat production worldwide. To overcome this rapid loss of resistance in varieties, we generated stable transgenic wheat plants expressing short interfering RNAs (siRNAs) targeting potentially vital genes of Puccinia striiformis f. sp. tritici (Pst). Protein kinase A (PKA) has been proved to play important roles in regulating the virulence of phytopathogenic fungi. PsCPK1, a PKA catalytic subunit gene from Pst, is highly induced at the early infection stage of Pst. The instantaneous silencing of PsCPK1 by barley stripe mosaic virus (BSMV)‐mediated host‐induced gene silencing (HIGS) results in a significant reduction in the length of infection hyphae and disease phenotype. These results indicate that PsCPK1 is an important pathogenicity factor by regulating Pst growth and development. Two transgenic lines expressing the RNA interference (RNAi) construct in a normally susceptible wheat cultivar displayed high levels of stable and consistent resistance to Pst throughout the T₃ to T₄ generations. The presence of the interfering RNAs in transgenic wheat plants was confirmed by northern blotting, and these RNAs were found to efficiently down‐regulate PsCPK1 expression in wheat. This study addresses important aspects for the development of fungal‐derived resistance through the expression of silencing constructs in host plants as a powerful strategy to control cereal rust diseases.     

Early molecular diagnosis and detection of Puccinia striiformis f. sp. tritici in China

Aims: Wheat stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most important foliar disease on wheat in China. Early molecular diagnosis and detection of stripe rust will provide a useful aid to the accurate forecast and seasonal control of this destructive disease. Our objective was to develop PCR assays for the rapid identification and detection of P. striiformis. Methods and Results: The genomic DNA of P. striiformis and P. triticina were amplified by a pair of primers derived from conserved β‐tubulin gene sequence. A 235‐bp specific DNA fragment of P. striiformis was isolated and purified. Based on its sequence, another two primer sets were designed successfully to obtain new sequence‐characterized amplified region (SCAR) markers of P. striiformis, which could be amplified in all test isolates of P. striiformis, whereas no DNA fragment was obtained in other nontarget wheat pathogens. The detection limit of the primer set YR (f)/YR (r1) was 2·20 pg μl^?1. The new SCAR markers of P. striiformis can also be detected in Pst‐infected wheat leaves postinoculated for 2 days. Conclusions: Our assays are significantly faster than the conventional methods used in the identification of P. striiformis. Significance and Impact of the Study: Development of a simple, high‐throughput assay kit for the rapid diagnosis and detection of wheat stripe rust would be anticipated in a further study.     

小麦抗条锈病近等基因系感染条锈病后丁布含量变化

选用2套遗传背景不同的抗条锈病近等基因系作为供试寄主材料,研究了不同抗条锈病近等基因系丁布的含量及其在感病过程中丁布含量的动态变化.结果表明,在未受病菌侵染情况下,2套分别含有Yr2、Yr9和YrSpP基因的近等基因系（抗病系）与其轮回亲本Taichung 29、铭贤169(感病系)间丁布含量没有显著差异（P＞0.05）.接种条锈病菌后,感病系在病菌侵染初期丁布含量下降,而抗病系在病菌侵染初期丁布含量迅速大幅度上升.感染条锈病最终导致感病植株丁布含量比未接种的植株明显减少, 感病系的减少幅度明显高于抗病系.在整个病程中,抗病系丁布的含量始终高于感病系,表明接种条件下小麦植株体内丁布含量变化与小麦抗条锈近等基因系的抗性有关.     

TaADF4, an actin‐depolymerizing factor from wheat,is required for resistance to the stripe rust pathogen Puccinia striiformis f. sp. tritici

Actin filament assembly in plants is a dynamic process, requiring the activity of more than 75 actin‐binding proteins. Central to the regulation of filament assembly and stability is the activity of a conserved family of actin‐depolymerizing factors (ADFs), whose primarily function is to regulate the severing and depolymerization of actin filaments. In recent years, the activity of ADF proteins has been linked to a variety of cellular processes, including those associated with response to stress. Herein, a wheat ADF gene, TaADF4, was identified and characterized. TaADF4 encodes a 139‐amino‐acid protein containing five F‐actin‐binding sites and two G‐actin‐binding sites, and interacts with wheat (Triticum aestivum) Actin1 (TaACT1), in planta. Following treatment of wheat, separately, with jasmonic acid, abscisic acid or with the avirulent race, CYR23, of the stripe rust pathogen Puccinia striiformis f. sp. tritici, we observed a rapid induction in accumulation of TaADF4 mRNA. Interestingly, accumulation of TaADF4 mRNA was diminished in response to inoculation with a virulent race, CYR31. Silencing of TaADF4 resulted in enhanced susceptibility to CYR23, demonstrating a role for TaADF4 in defense signaling. Using a pharmacological‐based approach, coupled with an analysis of host response to pathogen infection, we observed that treatment of plants with the actin‐modifying agent latrunculin B enhanced resistance to CYR23, including increased production of reactive oxygen species and enhancement of localized hypersensitive cell death. Taken together, these data support the hypothesis that TaADF4 positively modulates plant immunity in wheat via the modulation of actin cytoskeletal organization.     

中国小麦条锈菌转主寄主小檗的鉴定

用萌发的小麦条锈菌冬孢子接种采自陕西省境内的陕西小檗、少齿小檗和长穗小檗,3种小檗均产生了性孢子器和锈孢子器。用人工接种小麦条锈菌冬孢子在陕西小檗上产生的锈孢子器接种小麦铭贤169产生了典型的条锈菌夏孢子堆症状。特异性PCR和DNA序列分析表明,人工接种产生于小檗上的锈孢子、接种锈孢子于小麦上产生的夏孢子堆与小麦条锈菌DNA的ITS区序列完全一致。更为重要的是,用采自田间受锈菌侵染的小檗叶片产生的锈孢子接种小麦铭贤169,经培养在小麦铭贤169叶片上产生了典型的条锈病症状。从而证实,在自然条件下,在中国,小檗不仅可作为小麦条锈菌的转主寄主,而且小麦条锈菌可在小檗上完成其有性繁殖过程。这一发现对进一步揭示我国小麦条锈菌高度的群体遗传多样性与毒性变异机理、完善小麦条锈病的防治策略具有十分重要的理论和实际意义。     

四川省小麦条锈菌群体遗传多样性的SSR分析

利用TP-M13-SSR自动荧光检测技术,对四川省小麦条锈菌群体遗传多样性水平进行了分析。研究结果表明,四川省小麦条锈菌群体遗传多样性比较丰富,地区之间存在明显的差异,川西北和四川盆地的种群遗传多样性相对较高,而四川西南部和四川东南部的种群遗传多样性较低。四川小麦条锈菌群体存在一定的遗传分化,地区间的遗传变异仅占14.92%,群体间的遗传变异占总变异的23.06%,群体内遗传变异占60.02%,遗传变异主要存在于群体内部。基因流和共享基因型从分子水平证实了四川小麦条锈菌在地区间的传播,且川西北和四川盆地之间的菌源交流最为广泛。     

小麦条锈菌PsNCS1基因的克隆及转录表达特征

摘要：【目的】克隆小麦条锈菌神经钙离子感应蛋白基因PsNCS1,分析其在病菌不同发育时期的表达水平。【方法】利用文库筛选和RT-PCR技术克隆PsNCS1的cDNA序列,采用生物信息学技术预测分析该基因编码蛋白的保守结构域及基本特性,构建系统发育树;运用实时荧光定量RT-PCR技术分析PsNCS1在病菌不同生长发育时期的表达水平。【结果】PsNCS1全长cDNA为1007 bp（GenBank登录号GU134621）,开放阅读框为573 bp,编码190个氨基酸,分子量为22.17 kDa, 等电点为4.     

Isolation of twelve microsatellite loci,using an enrichment protocol,in the phytopathogenic fungus Puccinia striiformis f.sp. tritici

We report the characterization of 12 microsatellite markers in the biotrophic fungus Puccinia striiformis f.sp. tritici, responsible for yellow rust on wheat. An enrichment protocol was used to isolate microsatellite loci, and polymorphism was explored with 96 isolates from natural populations collected from several French and Chinese locations. Eight primers (67%) showed cross‐amplification when tested with eight isolates of P. triticina.     

条形柄锈菌小麦专化型葡萄糖-6-磷酸脱氢酶基因PsG6PDH1的表达特征及功能分析

葡萄糖-6-磷酸脱氢酶是磷酸戊糖途径中的关键限速酶。基于已测序的条形柄锈菌小麦专化型基因组序列,利用RT-PCR方法克隆了该病菌葡萄糖-6-磷酸脱氢酶PsG6PDH1的全长cDNA序列（1 497bp）,编码498个氨基酸的蛋白。编码蛋白含有葡萄糖-6-磷酸脱氢酶的保守功能域。系统进化分析发现,PsG6PDH1与禾柄锈菌小麦专化型的G6PDH聚为一簇。qRT-PCR分析表明,PsG6PDH1在病菌侵染早期的表达明显上调,其中侵染24h时表达量最高,为对照夏孢子的30倍。将PsG6PDH1导入酿酒酵母G6PDH缺失突变体中成功表达,并表现出较强的葡萄糖-6-磷酸脱氢酶活性,明显酵母增强了菌株对过氧化氢的耐受性。由此推测,PsG6PDH1可能参与了条形柄锈菌小麦专化型在侵染寄主时抵御寄主的氧化胁迫反应。研究结果为进一步研究该病菌基础代谢及侵染机理奠定一定的理论基础。     

Development of a sequence‐characterized amplified region marker using inter‐simple sequence repeats for detection of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Early and accurate detection of the pathogens would facilitate effective control of the diseases. DNA‐based methods now provide essential tools for accurate plant disease diagnosis. In this study, inter‐simple sequence repeats (ISSR) technique has been successfully applied to develop a sequence‐characterized amplified region (SCAR) marker for diagnosis of stripe rust of wheat and detection of Pst. In this study, one fragment unique to Pst was identified by ISSR and then sequenced. Based on the specific fragment, a pair of SCAR primers (616AF/616AR) was designed to amplify a 299‐bp DNA fragment within the sequenced region. The primers can amplify a unique DNA fragment for all tested isolates of Pst but not for the other pathogens of wheat leaves and the uninfected leaves. The polymerase chain reaction (PCR) assay could detect as low as 0.1 ng of genomic DNA in a 25.0 μl PCR reaction mixture and detect the pathogen from asymptomatic wheat leaves inoculated with Pst under glasshouse conditions.     

A polysaccharide deacetylase from Puccinia striiformis f. sp. tritici is an important pathogenicity gene that suppresses plant immunity

The cell wall of filamentous fungi, comprised of chitin, polysaccharide and glycoproteins, maintains the integrity of hyphae and protect them from defence responses by potential host plants. Here, we report that one polysaccharide deacetylase of Puccinia striiformis f. sp. tritici (Pst), Pst_13661, suppresses Bax‐induced cell death in plants and Pst_13661 is highly induced during early stages of the interaction between wheat and Pst. Importantly, the transgenic wheat expressing the RNA interference (RNAi) construct of Pst_13661 exhibits high resistance to major Pst epidemic races CYR31, CYR32 and CYR33 by inhibiting growth and development of Pst, indicating that Pst_13661 is an available pathogenicity factor and is a potential target for generating broad‐spectrum resistance breeding material of wheat. It forms a homo‐polymer and has high affinity for chitin and germ tubes of Pst compared with the control. Besides, Pst_13661 suppresses chitin‐induced plant defence in plants. Hence, we infer that Pst_13661 may modify the fungal cell wall to prevent recognition by apoplastic surveillance systems in plants. This study opens new approaches for developing durable disease‐resistant germplasm by disturbing the growth and development of fungi and develops novel strategies to control crop diseases.     

Isolation and characterisation of cDNA encoding a wheat heavy metal‐associated isoprenylated protein involved in stress responses

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal‐associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy‐terminal end. To investigate the roles of wheat heavy metal‐associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal‐associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real‐time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up‐regulated by ABA exposure or wounding. Additionally, TaHIPP1 over‐expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu²⁺ and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1‐GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus‐induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.     

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici,the causal agent of yellow rust disease

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation^* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (R_m) values ranging from 0.11 to 0.64 in the resolving gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes

The tomato I‐3 and I‐7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I‐3 has been identified previously on chromosome 7 and encodes an S‐receptor‐like kinase, but little is known about I‐7. Molecular markers have been developed for the marker‐assisted breeding of I‐3, but none are available for I‐7. We used an RNA‐seq and single nucleotide polymorphism (SNP) analysis approach to map I‐7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I‐7 as a gene encoding a leucine‐rich repeat receptor‐like protein (LRR‐RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I‐7, like many other LRR‐RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I‐7 gene for Fol resistance, we found that I‐7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I‐7‐mediated resistance, unlike I‐2‐ or I‐3‐mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1‐independent pathway for resistance activation. The identification of I‐7 has allowed us to develop molecular markers for marker‐assisted breeding of both genes currently known to confer Fol race 3 resistance (I‐3 and I‐7). Given that I‐7‐mediated resistance is not suppressed by Avr1, I‐7 may be a useful addition to I‐3 in the tomato breeder's toolbox.     

Systemic acquired resistance in Cavendish banana induced by infection with an incompatible strain of Fusarium oxysporum f. sp. cubense

Fusarium wilt of banana is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). The fact that there are no economically viable biological, chemical, or cultural measures of controlling the disease in an infected field leads to search for alternative strategies involving activation of the plant's innate defense system. The mechanisms underlying systemic acquired resistance (SAR) are much less understood in monocots than in dicots. Since systemic protection of plants by attenuated or avirulent pathogens is a typical SAR response, the establishment of a biologically induced SAR model in banana is helpful to investigate the mechanism of SAR to Fusarium wilt. This paper described one such model using incompatible Foc race 1 to induce resistance against Foc tropical race 4 in an in vitro pathosystem. Consistent with the observation that the SAR provided the highest level of protection when the time interval between primary infection and challenge inoculation was 10 d, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL, EC 4.3.1.5), peroxidase (POD, EC 1.11.1.7), polyphenol oxidase (PPO, EC 1.14.18.1), and superoxide dismutase (SOD, EC 1.15.1.1) in systemic tissues also reached the maximum level and were 2.00–2.43 times higher than that of the corresponding controls on the tenth day. The total salicylic acid (SA) content in roots of banana plantlets increased from about 1 to more than 5 μg g⁻¹ FW after the second leaf being inoculated with Foc race 1. The systemic up-regulation of MaNPR1A and MaNPR1B was followed by the second up-regulation of PR-1 and PR-3. Although SA and jasmonic acid (JA)/ethylene (ET) signaling are mostly antagonistic, systemic expression of PR genes regulated by different signaling pathways were simultaneously up-regulated after primary infection, indicating that both pathways are involved in the activation of the SAR.