Study On The Viromes Of Weed Plants And Sterile Rice Plants In Rice Fields

Rice is a major cereal crop for half of the global population.In addition to rice plants,various weed plant species grew in rice fields.Weed plants compete with rice plants for space resources,soil nutrients and light resources,they also serve as hosts for rice pathogens and insect pests.To date,many viruses were identified and isolated from rice plants.However,few viruses were reported in weed plants in rice fields.Rice plants are highly susceptible to rice virus infection and showed different symptoms.However,little is known about virus infection in rice plants that showed sterile symptoms.In this study,various weed plants were collected from different rice cultivation regions,and sterile rice plants infested by brown planthopper(BPH)were also collected.Using metatranscriptomic sequencing,we revealed viral diversity of the weed plants and the sterile rice plants.The results are as follows:1.29 weed plant samples,belonging to 24 weed plant species that were clustered within 13 families of plant species,were collected from major rice-producing regions in China(Hangzhou in Zhejiang province,Sanya in Hainan province,Guiyang in Guizhou province,and Xiaogan in Hubei province).Most of them were the members of the poaceae family.29 sequencing libraries were constructed for highthroughout sequencing.Using metatranscriptomic deep sequencing,sequence read assembly and similarity search,we identified 226 viruses including 310 viral genome sequences from the collected weed samples,and their genomes were sequenced to complete or near-complete length.According to criterion of the International Committee on Taxonomy of Viruses,14 were known virus species and 212 were newly discovered virus species,the identified viruses were clustered within 18 single positivestranded RNA virus families,3 single negative-stranded RNA virus families,and 1 double-stranded RNA virus families.Among them,67% of RNA virus genomes were detected from weed plant samples in Sanya,while only 8%,9% and 16% of RNA virus genomes were identified from weed plant samples in Hangzhou,Xiaogan and Guiyang,respectively.2.Phylogenetic analysis showed that most of the newly identified viruses filled in phylogenetic gaps between existing virus genera and families,and novel viruses described in this study greatly expanded the single positive-stranded RNA viruses(Virgaviridae,Marnaviridae,Dicistroviridae,Astroviridae,Nodaviridae,Solemoviridae,Tombusviridae,Tymoviridae,Botourmiaviridae,Mitoviridae,Narnaviridae,Fusariviridae,Atkinsviridae,Duinviridae,Fiersviridae,Solspiviridae,and Steitzviridae),single negativestranded RNA viruses(Peribunyaviridae,Phenuiviridae,and Leishbuviridae)and double-stranded RNA viruses(Partitiviridae).In the order Bunyavirales,21 newly identified viruses did not be clustered within current classified families,they were a large monophyletic group and a small monophyletic group,and we named the potential new virus family “Alphabunyaviridae” and “Betabunyaviridae”,respectively.3.Among the novel viruses,61 were clustered within the families or genera of fungal virus species(Mitoviridae,Narnaviridae,Fusariviridae,Magoulivirus,and Scleroulivirus),accounting for 30% of the total identified viruses.79 were grouped within the families or genera of bacteriophage virus species(Atkinsviridae,Duinviridae,Fiersviridae,Solspiviridae,and Steitzviridae),accounting for 35% of the total identified viruses.some novel viruses were clustered within the families or genera of several plant virus species or invertebrate-specific viruses.Of the identified viruses,31 were detected in all the four sampling regions,6 were identified from the different or the same collected samples in two or three regions,confirming that many viruses are widely distributed in rice fields in different regions.4.Using a backcross strategy,we found that integrating a dominant brown planthopper(BPH)resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance.However,when Bph3-carrying backcross rice plants are infested by BPH,these BPHresistant lines exhibit sterile characteristics.We used metatranscriptomic sequencing to identify 13 RNA viruses from the sterile rice plants,including 8 novel viruses and 5 known viruses,they were clustered within seven virus families(Phasmaviridae,Peribunyaviridae,Picornaviridae,Tombusviridae,Nodaviridae,Totiviridae,and Reoviridae)and 1 unclassified virus family of the order Mononegavirales.5.Small RNA sequencing analysis were performed on sterile rice plants infested by brown planthopper,the results showed that abundant small RNAs were identified for rice tombus-like virus 1and rice ragged stunt virus in Rby1-21 sample.Similarly,abundant small RNAs were also identified for rice tombus-like virus 1,rice ragged stunt virus,and rice peribunya-like virus in Rby2-45 sample.We analyzed all 18-30 nt small RNAs from the identified viruses in BPH-infested Rby1-21 and Rby2-45,and found that the virus-derived small interfering RNAs produced by Dicer-like proteins were mainly 21-nt and 22-nt.The results confirmed that virus infection triggered RNAi antiviral immune response in rice plants.In this study,high-throughput sequencing technology was used to identify virus genomes in weed plants and sterile rice plants infested by BPH,and we identified a total of 239 virus species from the collected plant samples.In addition,our results also confirmed that mixed virus infection may result in rice sterility.Together,the viral genetic and phenotypic diversity described here provide a new perspective on fundamental questions about viral origins,diversity,host range,genome evolution,and rice viral disease control.