Dynamics and Characterization of Small RNAs of Virus and Host Origin from Wheat Streak Mosaic Virus- and/or Triticum Mosaic Virus-infected Susceptible and Temperature-Sensitive Resistant Wheat Cultivars. Triticum aestivum

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are type members of Tritimovirus and Poacevirus genera, respectively, in the family Potyviridae, and are transmitted by wheat curl mites. Co-infection of these two viruses causes synergistic interaction with increased virus accumulation and disease severity in wheat. In this study, we examined the effects of synergistic interaction between WSMV and TriMV on endogenous small (s) RNAs and virus-specific small interfering RNAs (vsiRNAs) in susceptible (Arapahoe) and temperature-sensitive resistant (Mace) wheat cultivars at 27ºC and 18ºC. Single- and double-infections in wheat caused a shift in the profile of endogenous sRNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Additionally, we report high-resolution vsiRNA maps of WSMV and TriMV in singly- and doubly-infected wheat cultivars Arapahoe and Mace at 18ºC and 27ºC. Massive amounts of 21 and 22 nt vsiRNA reads were accumulated in Arapahoe at both temperatures and in Mace at 27ºC but not at 18ºC. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27ºC, although some regions of genomic RNAs serve as hot-spots with an excessive number of vsiRNAs. The positions of vsiRNA peaks were conserved among wheat cultivars Arapahoe and Mace, suggesting that Dicer-like enzymes of susceptible and resistant wheat cultivars are similarly accessed the genomic RNAs of WSMV and TriMV. Additionally, several cold-spot regions were found in the genomes of TriMV and WSMV with no or a few vsiRNAs, indicating that certain regions of WSMV and TriMV genomes are not accessible to Dicer-like enzymes. The high-resolution map of endogenous and vsiRNAs from wheat cultivars synergistically infected with WSMV and TriMV at two temperature regimens form a foundation for understanding the virus-host interactions, effect of synergistic interactions on host defense mechanisms, and virus resistance mechanisms in wheat. Overall design: Small RNA was sequenced from two wheat cultivars (Mace and Araphahoe), at two temperatures 18ºC and 27ºC, for healthy (control/uninfected), infected with wheat streak mosaic virus (WSMV), infected with Triticum mosaic virus (TriMV), and a double-infecttion of WSMV and TriMV.

小麦条纹花叶病毒（Wheat streak mosaic virus, WSMV）与小麦镶嵌病毒（Triticum mosaic virus, TriMV）分别为马铃薯Y病毒科（Potyviridae）下Tritimovirus属和Poacevirus属的模式种，二者均由小麦卷叶螨传播。两种病毒的共侵染可在小麦中引发协同互作，导致病毒积累量升高、病害严重程度加剧。本研究以感病小麦品种Arapahoe和温敏抗病小麦品种Mace为材料，探究了27℃与18℃条件下，WSMV与TriMV之间的协同互作对其体内内源小RNA（endogenous small RNAs, sRNAs）以及病毒特异性小干扰RNA（virus-specific small interfering RNAs, vsiRNAs）的影响。小麦单侵染与双侵染处理均可导致内源sRNA的分布谱发生偏移：健康植株中占比最高的24 nt小RNA，在侵染后的小麦中转变为以21 nt小RNA为主。此外，本研究还绘制了18℃与27℃条件下，单侵染及双侵染WSMV或TriMV的Arapahoe与Mace小麦体内的高分辨率vsiRNA图谱。在两种温度条件下的Arapahoe，以及27℃下的Mace体内，均积累了大量21 nt与22 nt的vsiRNA读段，但18℃下的Mace未检测到此类富集。正义链与反义链vsiRNA在两种温度下的Arapahoe体内，以及27℃下的Mace体内均广泛分布于病毒基因组RNA，尽管部分基因组区域存在vsiRNA富集的热点区域。vsiRNA的峰位在Arapahoe与Mace两个品种中保守，表明感病与抗病小麦品种的类Dicer酶均可类似地靶向WSMV与TriMV的基因组RNA。此外，在TriMV与WSMV的基因组中发现了若干无或仅存在少量vsiRNA的冷点区域，提示WSMV与TriMV基因组的部分区域无法被类Dicer酶识别。在两种温度条件下被WSMV与TriMV协同侵染的小麦品种中，内源小RNA与vsiRNA的高分辨率图谱，为解析小麦中的病毒-宿主互作、协同互作对宿主防御机制的影响，以及小麦抗病机制奠定了研究基础。实验总体设计：本研究针对两个小麦品种（Mace与Arapahoe），在18℃与27℃两个温度条件下，分别设置健康对照（未侵染）、单侵染WSMV、单侵染TriMV以及WSMV与TriMV双侵染四组处理，对其体内的小RNA进行测序。