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Genome-wide characterization of Rice Black Streaked Dwarf Virus-responsive genes in rice

Background: Rice black streaked dwarf virus (RBSDV) is an important pathogen disease in rice and gramineous planting regions all over the world. The major phenotypes of RBSDV infected rice were dwarf with dark green leaves, which generally resulted in seriously loss of yield. The RBSDV known so far is preoperatively transmitted to rice in a persistent manner by small brown planthopper (SBPH), instead of transmitting to offspring through ovary. Results: To identify RBSDV responsive genes and to explore and clarify the molecular mechanisms involved in plantvirus interaction, digital gene expression profile (DGE) analysis was performed by high-throughput sequencing using wildtype (WT) and RBSDV infected rice leaves (IRL) as materials. A total of 165,975 and 165,940 unique tags were obtained in IRL and WT libraries, respectively. In comparison with the control, 896 differentially expressed genes (DEGs) were obtained, of which 500 DEGs were up regulated and 396 DEGs were down regulated. Functional analysis showed that DEGs mainly classified into10 groups, including metabolism, stress pathogen and defense, signal transduction, Transporter, transcription and post-transcription, cell structure and division etc. To further validate reliability and authenticity of the data, 10 DEGs were randomly picked and Real-time RT-PCR was carried out, and the expression trends of 7 genes were in line with the RNA-seq results. By searching the RBSDV related miRNA database of rice, 10 targeted genes of 6 significantly changed miRNAs were also identified in these DEGs. Conclusions: The data derived from RNA-seq were valid and credible. Through this research, a series of candidate RBSDV-responsive genes were obtained, and special signal transduction and metabolism pathways were built and pulled out in rice. This study provided further insight into the molecular mechanisms during compatible and incompatible interactions between viruses and their host plants. Copy rights: © This is an Open access article distributed under the terms of International License. Genome-wide characterization of Rice Black Streaked Dwarf Virus-responsive genes in rice Journal of Food Science & Technology (ISSN: 2472-6419) DOI: 10.25177/JFST.5.2.RA.615 Research


INTRODUCTION
Rice black streaked dwarf virus (RBSDV), which belongs to the genus Fijivirus within the family Reoviridae, could propagated and infected rice, barley, wheat, sorghum, maize and other host plants by planthopper laodelphax striatrllus in a persistent manner. The harmful influence of corresponding disease is reported to cause extensive and devastating crop losses in grain and forage yield, which ultimately leading serious economic loss in Asian countries such as China and Japan [1]. The infection of rice by RBSDV usually cause distant disease symptoms and growth abnormalities, such as severe dwarfing, more ineffective tillerings, poorly developed roots, rigidity leaf with almost no heading [2].
The whole genome of RBSDV consists of 10 doublestranded RNA segments (S1-S10) [3,4]. The complete nucleotide sequences of these segments have been determined and the functions of encoding proteins also have been preliminary researched. The mechanism by which many proteins are involved in plant virus interaction has also been studied. For instance, S7 contains two ORFs which encoding p7-1 and p7-2 proteins. Therein P7-1 may be disruption of the lignin biosynthesis and H 2 O 2 -dependent polymerization pathways, and P7-2 might involve in the -virus interaction [5,6]. S8 encodes core capsid protein, which may act as a nuclear transcriptional inhibitory factor in plant cells that participated in host cell gene expression regulation to make it suitable for virus propagation [1]. Although the biological functions of viral genome becoming increasingly clear, there were still no effective strategies to monitoring and control this disease as the insufficient data of the interactions between RBSDV and its host plants.
Microarray investigation revealed that various disease resistance genes, cell wall-related genes and development-related genes were altered in RBSDV infected maize [7]. Proteomic investigation also revealed that the dramatic changes of the basal metabolism processes in infected maize. Moreover, the demands for antioxidant enzymes, G-proteins, lipoxygenases and UDP-glucosyltransferase BX9 all increased in the virus infected plants, which indicated that they may participate in disease resistant response[2]. Using comparative proteomic analysis, 72 differently expressed proteins which belonged to 11 groups were detected in RBSDV permanently infected rice [8]. Using deep sequencing approach, the expression of 14 miRNAs in leave and 16 miRNAs in root were found significantly changed in RBSDV infected rice [9]. Genetic analysis also showed that the resistance of rice variety Tetep to RBSDV was controlled by quantitative trait locis (QTLs), and three new QTLs were also identified [10].
High-throughput sequencing technology has been used to researches of various virus infected plant species, such as Arabidopsis thaliana [11,12], Nicotiana [13], Zea may [14], potato [15] and rice [16]. As a model plant, the multiple genome and transcriptome databases and bioinformatical resources of rice are already available, which make it practicable to carry out high-throughput sequencing.
In this research, using WT and the RBSDV infected rice leaves (IRL) as materials, the genes expression profiles were investigated by RNA-seq, and the physiological and biochemical indexes were detected. Finally, about 896 DEGs were identified, and partial expression patterns were further verified. The expression patterns of several disease-related miRNAs targeted genes were also detected. The expression profiles or transcriptomes of the genes have provided effective clues for plants disease resistance breeding and germplasm innovation.

MATERIALS AND METHODS 2.1Plant materials
Epidemiological investigation of RBSDV was carried out in Jining, Shandong. As the production and quality of RBSDV susceptible rice variety "Zixiangnuo 2315" were heavily infected by RBSDV infection, ten RBSDV infected rice plants with symptoms were picked while the healthy strains were harvested as control locally (Figure 1). All of the rice samples were stored in -80℃ for further analysis. To verify whether the dwarf symptoms were caused by RBSDV infection, two pairs of specific PCR primers (supplement table 2) of published virus S9 and S10 were designed.

RNA extraction and cDNA libray construction
Total RNA of WT and IRL were extracted using Trizol reagent, and the crude extracts were treated with RNase-free DNaseⅠ(TaKaRa, Dalian, China). In order to reduce the individual error, the RNA of 3 independent strains of RBSDV infected or wild type plants were mixed as one sample. The total RNA concentration was examined with a spectrophotometer (Nanodrop ND-2000, ThermoFisher Scientific, Wilmington, DE, USA), and the RNA sample integrity was verified by a Bio-Analyzer 2100 (Agilent Technologies, Santa.Clara, USA). The cDNA libraries construction and Sequencing at BGI Tech Solutions Co., Ltd. (BGI Tech, Shenzhen, China).

Identification of differential expressed genes (DEGs)
Obtaining and screening the unambiguous clean tags, the number of unambiguous clean tags for each gene were calculated and then normalized to TPM (number of transcripts per million clean tags).
The DEGs between two samples were identified by a rigorous algorithm method. The "FDR (False Discovery Rate)≤0.001 and the absolute value of log2Ra-tio≥1" were used as the threshold to judge the significance of gene expression difference. The biological replication was analysis using Pearson correlation. According to the correlation results, DEGs were selected based on the NOISeq method of Q≥0.6 [17].

Gene Ontology (GO) and pathway enrichment analysis of DEGs
GO and pathway annotation and enrichment analysis were based on the Gene Ontology Database (http:// www.geneontology.org/) and KEGG pathway Database (http://genome.jp/kegg/).

Quantitative Real-time PCR (qRT-PCR) Analysis
The gene-specific primers were designed using pri-mer3.0 plus (supplement table 2). The qRT-PCR was performed using FastStart Universal SYBR Green Master (Roch, Shanghai, China) according to the manufacturer's instructions. Each reaction was performed refer to Liu Wei report [18]. The rice actin gene (LOC_Os03g50885) was used as internal control. Quantification of the relative changes in gene expressions were performed using the 2 -△△CT method as described by Livak and Schmittgen [19].

MicroRNA expression analysis
Refered to the data of RBSDV responsive miRNAtargeted genes [9], the putative target miRNAs of DEGs in this research were obtained.

Verification of virus infected rice strains
The RBSDV susceptible rice that sampling from Jining were preliminary screened according to Li Shuo(2015) report [20,21]. After been screened by specific primers of virus segments of S8 and S9 by RT-PCR, eight RBSDV infected rice strains with notable stunting symptoms and nine healthy strains were obtained (Supplement figure 1).
Considering RBSDV and rice stripe virus (RSV) all can be transmitted by planthopper laodelphax striatrllus simultaneously, the RSV specific primers (Supplement table 2) were also used to distinguish single virus infection or co-infection. The results showed that only one strain was detected to be infected with both RBSDV and RSV, other strains were all RBSDV single virus infection.

Tags identification and quantification
After library construction and RNA-seq analysis, total of 4,069,983 and 4,854,614 tags were obtained separately in IRL and WT. There about 3,662,063 and 4,559,710 clean tags were left after per-filter. Then, only tags with less than one copy remain in the library and were used for further analysis. Finally, 165,975 and 165,940 unique tags for the IRL and WT were obtained, respectively. All clean tags were mapped to the reference genes and genome of rice. There were 65,457 (39.45% of clean tags) unique tags in WT library and 57,703 (34.77% of clean tags) unique tags in IRL library, and the unique tags were mapped to 17,785 (26.81%) and 17424 (26.27%) of the reference genes, separately (Table 1). Meanwhile, 41,422 (24.96%) and 40,048 (24.13%) unique tags were mapped to reference genome (Table 1).

Gene expression analysis between WT and IRL libraries
About 896 DEGs were identified between WT and IRL libraries, among which 500 genes were up regulated and 396 genes were down regulated. The DEGs were mainly divided into 10 groups, among which more than 50% of DEGs were involved in stress and defense response and metabolic processes. Other DEGs were classified into hormone biosynthesis and signal transduction, Protein biosynthesis, cell structure and division ( Figure 3).
Considering the screening of the disease resistance genes and breeding of disease resistance rice varieties, the categories of photosynthesis-related DEGs, hormone biosynthesis and signal transduction related DEGs, pathogenesis-related DEGs, and other remarkable DEGs were analyzed emphatically.

Photosynthesis-related DEGs
Here totally 23 photosynthesis-related DEGs were down-regulated in RBSDV-infected rice. Among which 18 DEGs belong to photosystem II reaction center protein, chlorophy Ⅱ A-B binding protein, photosystem I reaction center protein, photosynthetic electron transport and F-type ATPase (Table 2). There were 5 DEGs that function in the Calvin-Benson cycle, including two fructose-1,6bisphosphatase, one phosphoribulokianse/Uridine kinase family protein, one ribulose bisphosphate carboxylase small chain, and one phosphoglycerate kinase protein (Table 2 and Table 3) .  1 based on Rice Genome Annotation Project database 2 Log 2 -based differential expression ratio Vol-5 Issue-2

Synthesis, metabolism and signal transduction related DEGs of plant hormones
Considering the dwarf and stunted phenotypes of RBSDV infected rice, multiple clues all indicated that RBSDV disease is closely related to the synthesis and metabolism of plant hormones [22][23][24]. The genes involved in corresponded processes and signal transduction pathways were focused.

Genes related to Gibberellins biosynthesis and signaling
Here three significantly DEGs of GA biosynthesis were identified. The expression decreased genes mainly included one ent-copaly1 diphosphate synthase (CPS) and two gibberellins 20 oxidase 2 (GA20ox 2). Meanwhile, the expression levels of four genes encoding GID1 were increased (Table 4), which known to be involved in GA signaling pathway.

Genes related to brassinolide biosynthesis and signaling
Two DEGs encoding cytochrome P450 were induced to express by RBSDV which identified to be involved in BRs biosynthesis. In the BRs signal transduction pathway, the BRASSONOSTEROID -INSENSITIVE 1 (BRI1), BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) and BSU1 were all found to be up-regulated under disease infected conditions (Table 5).

Other remarkable DEGs
Except the DGEs that classified into specific processes above, there still have 23 DEGs that present significant high expression levels, and 10 DEGs that showed decreased expression levels in RBSDV infected rice (log2Ratio IRL/WT>5) ( Supplement Table 1). Among which, there were 17 up-regulated genes and 7 down-regulated genes that have annotations in rice.
OsCML26, which reported to play important roles in the signal transduction and transcriptional regulation networks of Ca 2+ signaling in response to different abiotic stress, was induced to express significantly (log 2 Ratio=8.64) after RBSDV infected [33,34].
In the data, we also found two cell wall-related genes, one was POEI5-Pollen Ole e I allergen and extensin family protein precursor (LOC_Os10g05820.1), which is a key enzyme in the biosynthesis of lignin cell wall precursors, the expression level of this gene was down-regulated after RBSDV infected (log 2 Ratio=-8.03). The other was pectinesterase, which is a cell-wall-associated enzyme that facilitates plant cell wall modification and subsequent breakdown. The expression level of this gene was up-regulated in infected rice (log 2 Ratio=7.80).

Gene Ontology (GO) enrichment analysis of DEGs
In order to understand the functions of these DEGs, all of these genes were mapped to GO database and classified into different groups according to GO terms comparing to the genome background.
In the category of molecular function (MF) the GO terms of oxidoreductase activity and binding which including pattern binding, carbohydrate binding and ion binding were significantly overrepresented, other terms such as chitinase activity were also dominant. When looking into the category of biological process (BP), the terms of response to stress and response to biotic stimulus were found overrepresented in the IRL library, which were also found in other plants infected with pathogen. Other terms such as aminoglycan catabolic process, amine metabolic process and polysaccharide catabolic process were also dominant.

Verification of profiles of DEGs
To validate the expression profiles of DEGs, 10 DEGs were picked randomly and analyzed by qRT-PCR using rice actin as internal control. The results showed that except 3 genes, the variation trends of other 7 genes were consistent with RNA-seq results (Supplement figure 2).

MicroRNA expression analysis
There were 10 DEGs targeted by miR164, miR166, miR396, miR528, miR827, miRn11. Including NAC gene, ozone-responsive apoplastic protein, OsSPX-MFS1, and so on (Table 7 ). But the expression of DEGs was notable unless ozone-responsive apoplastic protein. And the expression levels of these miRNAs will be further analyzed by qRT-PCR.

Effects of RBSDV on photosynthetic rate and energy metabolism
Plant infected with RBSDV usually induces photosynthetic rate decrease. The long-term RBSDV accumulation in rice lead to the decrease of photosynthetic parameters, including the net photosynthetic rate (Pn), the stomatal conductance (Cond), the intercellular CO 2 concentration (Ci), and the transpiration speed (Tr). Some related proteins such as chlorophyII a/b binding proteins, Rubisco activase small isoform precursor and calvin cycle related proteins were also and psbW (LOC_Os03g21560.1). The psbA encode photosynthetic reaction center protein D1, and the psbP encode subunit of the oxygen evolving complex (OEC). The decrease of the protein products of these two genes will affect the assembly and/or stability of the PSII-LHCII supercomplexes in the gram regions of the thylakoid membrane [35][36][37]. The PsbY protein was reported to take part in the cyclic electron flow around PSII in order to protect PSII against photoinhibition, and also could stabilize the binding of PsbE and PsbF to the heme group by providing a shelter of Cyt b 559 from oxidizing compounds [38]. The psb27 was known to be essential for efficient recovery of the photodamaged PSII. In Arabidopsis, under highlight-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant [39,40]. Taken together; the decrease of these

SIFT DESK
photosynthetic genes will affect the stable of PSII complex and the recovery of the photodamaged PSII. Five Chlorophyll A-B binding protein genes were down-regulated in RBSDV infected rice. Considering chlorophyll A-B binding protein is a component of the light-harvesting complex of photosynthesis II (LHC II), down regulation of these genes may inhibit LHC II function and further affect the photosynthetic process.
In the photosystem I, there were 4 DEGs including psa D (LOC_Os08g44680.1), psaG (LOC_Os09g30340.1), psaN (LOC_Os12g08770.1) and psaO (LOC_Os04g33830.1). In view of the functions of proteins encoded by these genes, the down regulation of these genes may affect stable and activity of PSI, and influence the energy balance between photosystems [41][42][43]. The reduction of plastocyanin (PC) and ferredoxin--NADP reductase(FNR) may inhibit the electron transport from PS II to PSI. RBSDV infection also inhibited the carbon fixation of plants by decreasing the expressions of 5 DEGs which encoding Calvin cycle enzyme.
Taken together, the PSI, PSII and electron transport were infected in BSDV infected rice, and thereby infected the photosynthesis efficiency and energy accumulation, which further results in abnormal growth, dwarfing and fruitless symptoms of infected plants.

The relationship between dwarfing symptoms and hormones
Dwarfism is a typical characteristic symptom of RBSDV infected plants. Considering phytohormones play multiple roles in regulation growth and development of plants, their variations in RBSDV infected rice were analyzed in depth.
Gibberellins (GA) are a group of tetracyclic diterpenoid compounds, which essential for many processes of plant growth and development [44][45][46]. The loss of function in GA synthesis and signaling resulted in dwarfism in rice and Arabidopsis plants, and transgenic plant expressing genes for GA degradation showed the dwarfism [47][48][49]. In RBSDV infected rice the GA content reduces significantly [50]. In this research, partial DEGs in GA biosynthesis were decreased and in GA signaling pathway were increased, suggested that RBSDV infection might inhibite the accumulation of gibberellins, but activate GA signaling transduction in rice.
Brassinosteroid (BR) is other important phytohormone in plants. Brassinosteroids involve in various physiological processes including plant growth, development and immunity [51].In rice , a low concentration of BL (10 -9 M) could slightly enhance leaf sheath and root length, but it will inhibit leaf sheaths and roots growth when the contents were accumulated over certain levels, which resulted in decreased seeding height and root length [52]. Consistent with this, mutant or transgenic plants with excessive enhanced BR levels or BR signaling usually have reduced height to a certain degree in rice. In our data, two DEGs in BR biosynthesis were up-regulated significantly under virus infection, and this give us a hint that it may be caused by over accumulation of BR that lead to the dwarfism of RBSDV infected rice. The detailed mechanisms were still under investigation.

The roles of PRs in RBSDV infected rice
By now, many theoretical researches on the effects of plant PR genes have mainly focused on plant-fungi/ bacteria interactions [53]. Some research found that the induced patterns of the PRs were quite different in virus infected rice, hinting the divergent defense mechanisms during defense response. In this research, 75 PRs that 61 up-regulated and 14 downregulated were identified in the highly RBSDV susceptible rice Zixiangnuo2315. The PR have been classified into 17 groups (PR1-PR17) based on their amino acid sequences and biological functions [54].In our data, These 75 PRs belong to eleven PR subfamilies, and the DEGs of PR1, PR14 and PR16 changed significantly in this research [54]. Especially seven DEGs of PR1 (including OsPR1a and Os-PR1b) were dramatically induced to express about 10 to 3000 folds, which displayed vital roles in RBSDV and host interaction [55,56]. Here ten PR genes belong to PR14 subfamily, which further defined as

SIFT DESK
LTP genes were also changed, among which 6 genes were down-regulated about 3 to 285 folds and 4 genes were up-regulated about 3 to 20 folds [57]. As reported that LTP genes such as OsC6 may participate in development regulation of pollen, the phenotypes of no heading or sterility of RBSDV infected rice may association with down-regulation of LTPs [58].