Comparative analysis of transcripts associated with all-stage resistance to stripe rust in wheat

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a destructive disease of wheat worldwide. Genetic resistance is the preferred method for controlling stripe rust, of which two major types are race-specific and race non-specific resistance. Race-specific resistance includes the qualitatively inherited all-stage resistance, controlled by single major resistance (R) genes. Conversely, adult-plant resistance is race non-specific, inherited quantitatively, and is durable. Previously, we characterized the gene expression signatures involved in Yr5-controlled all-stage resistance and Yr39-controlled adult-plant resistance using the Affymetrix Wheat GeneChip. For this study, we designed and constructed custom oligonucleotide microarrays containing probes for the 116 and 207 transcripts that we had found important for the Yr5 and Yr39 resistance responses, respectively. We used this custom microarray to profile the resistance responses of eight wheat genotypes with all-stage resistance (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15, and Yr17). The aim of this analysis was to identify common and unique gene expression signatures involved in race-specific resistance accross genotypes, which were used to infer information regarding the general pathways involved in all-stage resistance. Keywords: Stress response Overall design: Eight wheat genotypes with defined single gene all-stage resistance to particular stripe rust (Pst) isolates were selected for this study, as well as the Pst susceptible genotype ‘Avocet Susceptible’ (AVS). Near Isogenic Lines (NILs) for each of eight Yr resistance genes (Yr1, Yr5, Yr7, Yr8, Yr9, Yr10, Yr15 and Yr17) were developed at the Plant Breeding Institute, Sydney, Australia, by backcrossing the Yr gene donors with the recurrent susceptible spring wheat genotype (Triticum aestivum L.) AVS six times (AVS*6/Yr*) and selecting for the appropriate resistance at each generation. Both virulent and avirulent isolates of Pst were selected for each NIL, except for Yr15, for which no virulent isolate is currently known. Care was taken to select the fewest isolates needed to satisfy the spectrum of virulence/avirulence required, resulting in the use of six isolates identified as PST-17, PST-21, PST-43, PST-45, PST-78 and PST-AUS. Each isolate was selected and maintained on susceptible genotypes. For each of three biological replications, individual genotypes were planted in separate 25 X 42.5-cm flats using a potting mix (6 peat moss: 4 vermiculite with lime: 3 sand: 3 commercial potting mix: 2 perlite: 1.7 g/L lime: 3.3 g/L Osmocote: 2.2 g/L ammonium nitrate). Each flat consisted of three rows of six seedlings, with rows randomly assigned one of two harvest times (24 and 48 h post-inoculation). Seedlings from the 3rd row were used to monitor the expected disease responses to inoculation. Seedlings were grown to the second leaf stage (Feekes 1.2, emergence with second leaf unfolded, ~10 days after planting) in a greenhouse with a diurnal temperature cycle of 10ºC (2:00 am) to 25ºC (2:00 pm) and a 16 h light/8 h dark cycle. Inoculation was performed by misting the plants with sterile water and applying a 1:20 urediniospore:talc mixture to leaves with a sterile brush. Talc was used to aid in the spread and adhesion of spores over leaf surfaces. Control flats were treated the same way except for the absence of spores in the talc. All treatments for each biological replication were performed at 9 am Pacific Standard Time. To promote spore germination, all flats were transferred to a dew chamber (100% RH) operating at 10ºC in the dark for 24 h, before being placed in a growth chamber with a diurnal temperature cycle of 4ºC (2:00 am) to 20ºC (2:00 pm) and a 16 h light/8 h dark cycle. Rows of plants were harvested from all flats at the assigned times for RNA extraction.

条纹锈病（Stripe rust），由 Puccinia striiformis f. sp. tritici 引发，是全球范围内小麦作物的一种毁灭性病害。遗传抗性是控制条纹锈病的首选方法，其中主要包括谱系特异性抗性和非谱系特异性抗性。谱系特异性抗性包括定性遗传的全程抗性，由单个主要抗性（R）基因控制。反之，成株抗性为非谱系特异性，呈数量遗传，且具有持久性。先前，我们利用 Affymetrix 小麦基因芯片对由 Yr5 控制的全程抗性和 Yr39 控制的成株抗性所涉及的基因表达特征进行了表征。在本研究中，我们设计并构建了包含针对我们发现的与 Yr5 和 Yr39 抗性反应相关的 116 和 207 个转录本探针的定制寡核苷酸微阵列。我们使用该定制微阵列对具有全程抗性的八个小麦基因型（Yr1、Yr5、Yr7、Yr8、Yr9、Yr10、Yr15 和 Yr17）的抗性反应进行了分析。本分析的目的是识别跨基因型的谱系特异性抗性中共同和独特的基因表达特征，并据此推断涉及全程抗性的通用途径。关键词：应激反应，总体设计：本研究选取了具有特定条纹锈病（Pst）分离株定义的单基因全程抗性的八个小麦基因型，以及易感基因型‘Avocet Susceptible’（AVS）。在澳大利亚悉尼的植物育种研究所，通过将 Yr 基因供体与轮回易感春小麦基因型（Triticum aestivum L.）AVS 进行六次回交（AVS*6/Yr*）并选择每一代中的适宜抗性，开发了八个 Yr 抗性基因（Yr1、Yr5、Yr7、Yr8、Yr9、Yr10、Yr15 和 Yr17）的近等基因系（NILs）。对于每个 NIL，除了 Yr15（目前尚未发现致病分离株）外，都选择了致病性和非致病性分离株。我们精心选择了所需的最少分离株，以满足所需的致病性/非致病性范围，最终使用了 PST-17、PST-21、PST-43、PST-45、PST-78 和 PST-AUS 六个分离株。每个分离株均选自易感基因型进行培养。在每个生物重复中，单个基因型分别种植在独立的 25 X 42.5-cm 花盆中，使用混合土壤（6 苔藓：4 珍珠岩：3 沙子：3 商业土壤：2 珍珠岩：1.7 g/L 石灰：3.3 g/L Osmocote：2.2 g/L 硝酸铵）。每个花盆包含三行六株幼苗，行随机分配两种收获时间（感染后 24 和 48 小时）。第三行的幼苗用于监测预期对感染的疾病反应。幼苗在温室中生长至第二叶期（Feekes 1.2，第二叶展开后出现，种植后约 10 天），温室的昼夜温度循环为 10ºC（凌晨 2 点）至 25ºC（下午 2 点），光照/黑暗周期为 16 小时/8 小时。感染通过喷洒无菌水和用无菌刷将 1:20 urediniospore:talc 混合物涂抹在叶片上完成。滑石粉用于帮助孢子在叶片表面扩散和粘附。对照组花盆的处理方式相同，但滑石粉中不含孢子。每个生物重复的所有处理都在太平洋标准时间上午 9 点进行。为了促进孢子发芽，所有花盆均转移到湿度为 100% 的冷室（10ºC，黑暗）中 24 小时，然后再放置在具有昼夜温度循环为 4ºC（凌晨 2 点）至 20ºC（下午 2 点）和 16 小时光照/8 小时黑暗周期的生长室内。在指定的时间从所有花盆中收获植株行以提取 RNA。