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

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.

小麦线条花叶病毒（Wheat streak mosaic virus, WSMV）和小麦花叶病毒（Triticum mosaic virus, TriMV）分别是马铃薯Y病毒科（Potyviridae）下小麦病毒属（Tritimovirus）和禾本科病毒属（Poacevirus）的模式成员，由小麦曲叶螨传播。这两种病毒的共感染会引发协同互作，导致小麦体内病毒积累量增加且病害严重度上升。本研究探讨了WSMV与TriMV的协同互作对易感小麦品种阿拉帕霍（Arapahoe）和温度敏感抗性品种梅斯（Mace）在27℃和18℃条件下内源小RNA（small RNA, sRNAs）及病毒特异性小干扰RNA（virus-specific small interfering RNA, vsiRNAs）的影响。小麦被单一或两种病毒感染后，内源sRNA的谱图发生变化——健康植株中占主导的24 nt sRNA转变为感染植株中的21 nt sRNA。此外，我们报道了WSMV和TriMV在单一及共感染的阿拉帕霍和梅斯小麦品种中于18℃和27℃条件下的高分辨率vsiRNA图谱。阿拉帕霍在两种温度下、梅斯在27℃下均积累了大量21 nt和22 nt的vsiRNA reads，但梅斯在18℃下无此积累。在两种温度条件下的阿拉帕霍及27℃下的梅斯中，正义链和反义链vsiRNA分布于整个基因组RNA，尽管基因组RNA的某些区域是vsiRNA过度富集的热点区域。vsiRNA峰的位置在阿拉帕霍和梅斯品种间具有保守性，表明易感和抗性小麦品种的Dicer样酶对WSMV和TriMV基因组RNA的访问模式相似。此外，在TriMV和WSMV基因组中发现了若干冷点区域（无或极少vsiRNA），表明这两种病毒基因组的特定区域无法被Dicer样酶访问。在两种温度条件下被WSMV和TriMV协同感染的小麦品种中，内源sRNA和vsiRNA的高分辨率图谱为理解病毒-宿主互作、协同互作对宿主防御机制的影响及小麦抗病毒机制奠定了基础。
总体设计：从小麦品种梅斯（Mace）和阿拉帕霍（Arapahoe）中测序小RNA，涵盖两种温度条件（18℃和27℃）及四种处理：健康对照（未感染）、WSMV感染、TriMV感染、WSMV与TriMV共感染。