草莓和柑橘中新类病毒的发现及其致病机制研究

Viroids represent a unique class of pathogens that are circular RNA molecules of a few hundreds of nucleotides in length. Viroids cause important diseases in potato, tomato, citrus, avocado and other crops and spread mainly by vegetative propagation, mechanical contamination and through pollen and seed. However, identification of new viroids is technically demanding since it requires purification of the naked viroid RNA that occurs generally in low concentrations in infected plants and thus is pursued only after an extensive search has failed to identify a viral or bacterial pathogen for a disease. This probably explains why less than 40 viroids in total have been identified following the discovery of the first viroid more than 40 years ago and why viroids are not yet found in many crops. .In plants, RNA silencing has been proven to be a major defense mechanism against viroid infection. The double stranded RNAs of viriods are recognized by Dicer-like enzyme and cleaved into 21～24 nt siRNAs. Our deep sequencing has revealed that those small interfering RNAs (siRNAs) produced in infected host cells overlap each other in sequences. Therefore, We have developed a new computational algorithm to identify circular RNA molecules in a host in a homology-independent manner. According to the algorithm, the small RNAs will be filltered and assembled by PFOR program and only representative sequences of viroid candidates will be yielded for further analysis..We propose here to test the hypothesis that many new viroids with little homology to the known viroids occur in nature, cause diseases in important crops, and escape detection because of technological limitation. We will focus on viroid discovery in strawberry and citrus because they are propagated vegetatively, have uncharacterized diseases and disease complexes. The total RNAs of these samples will be isolated and small RNAs will be enriched further for constrtuction of small RNA libraries. The small RNA libraries will be sequenced to high coverage by next generation sequencer. Then reads from each library will be used to identify new viroid candidates. RT-PCR and northern blot will be performed to verify the presences of viroids in samples. Infectivity and pathogenesis of the identified viroid candidates from strawberry and citrus will be verified by constructing the infectious cDNA clones. We will determine if the newly identified viroids share sequence, structural and functional domain similarities and can be grouped into new viroid families. The proposed project can potentially identify diverse viroids pathogenic to berry and citrus important to the China agriculture.

类病毒是一类能够引发许多农作物疾病的重要病原体。我们新近发展了一种利用类病毒小RNA相互重叠的特征来发现新类病毒的方法，能发现与已知类病毒没有序列相似性的全新类病毒。我们的方法克服了传统发现新类病毒方法的技术瓶颈，将可能促进在许多物种中发现新类病毒。我们计划以繁殖性生长且具有未定性的疾病或疾病复合体的草莓和柑橘为研究对象，构建这些样本的小RNA文库，通过深度测序获取小RNA的序列，然后通过我们发展的软件PFOR发现候选新类病毒，并应用实验手段验证候选新类病毒的存在。我们还将以在草莓和柑橘中新发现的候选类病毒和前期工作中已发现的4个柑橘候选类病毒为研究对象，构建它们的侵染性克隆，研究它们的自我复制和致病机制。通过本课题研究，我们将从草莓和柑橘样本中发现全新的类病毒，并完成它们病毒致病机制的初步分析，对我国草莓和柑橘植物疫病的诊断和防治具有重要意义；同时为研究类病毒的起源进化机制提供研究基础。

RNA沉默是植物抵御病毒和类病毒感染的一种主要的防御机制，并在细胞内形成来源于病毒或类病毒来源的小RNA。这些小RNA相互重叠，能通过生物信息学方法组装成较长的序列，从而便于后续的分析来鉴定发现新的病毒和类病毒。本课题在项目组成员前期开发的用于鉴定植物病毒的vdSAR方法和鉴定植物类病毒PFOR方法基础上，进一步优化这些软件，并应用这些方法策略来分析未定性的疾病或疾病复合体的植物样本，来鉴定发现新的病毒和类病毒。主要研究内容和研究成果包括：（1）针对PFOR软件分析耗时过长的问题，采用C++语言中OpenMP多线程并行处理方案对PFOR进行重新编程，并植入SLS程序，使得PFOR2不仅在运行速度上大大提高，而且不仅局限于分析小RNA数据，也适合对RNAseq数据进行分析处理。（2）以100多年树龄的葡萄树为研究对象，分析发现4个已知类病毒HpSVd、GYSVd-1、GYSVd-2和AGVd，并且发现一个长度为328nt新类病毒GLVd。通过实验验证了GLVd环状性质及其在葡萄树中具有自我复制的能力。系统发育分析表明GLVd与CVd-VI的进化关系最近，是隶属于苹果锈果类病毒属的新成员。 (3) 以具有苹果锈果病症状的苹果树为研究对象，分析发现该样本不仅含有已知类病毒ASSVd，同时还发现一个长度为434nt的全新的环状RNA。 通过实验验证了该RNA的其环状特性和其核酶的体外自切活性，但目前未能验证其侵染性活性，因此这个环状RNA暂被命名为AHVd-like RNA。(4) 通过分析锦紫苏样本，鉴定了两个已知类病毒CBVd1和CBVd5，并发现一个二级结构为杆状的全新的候选类病毒序列。(5) 通过分析表现黄化病的海南槟榔样本，鉴定了隶属Velarivirus属的新病毒APV1。（6）通过分析具有黄化症状的草莓样本，鉴定了属于甲型线形病毒科的新病毒SMYEaV1。 (7) 对利用高通量测序和生物信息学分析鉴定发现植物病毒和类病毒文献进行了系统总结，并在Annu Rev Phytopathol 杂志上发表了该综述。通过本课题研究，我们优化了发现植物病毒和类病毒的方法，发现了一些重要植物的新病毒和类病毒，并在Plos pathogens、Annu Rev Phytopathol、Arch Virol等杂志上发表了6篇SCI论文。