Identification, Characterization and Full-Length Sequence Analysis of a Novel Polerovirus Associated with Wheat Leaf Yellowing Disease

Abstract

To identify the pathogens responsible for leaf yellowing symptoms on wheat samples collected from Jinan, China, we tested for the presence of three known barley/wheat yellow dwarf viruses (BYDV-GAV, -PAV, WYDV-GPV) (most likely pathogens) using RT-PCR. A sample that tested negative for the three viruses was selected for small RNA sequencing. Twenty-five million sequences were generated, among which 5% were of viral origin. A novel polerovirus was discovered and temporarily named wheat leaf yellowing-associated virus (WLYaV). The full genome of WLYaV corresponds to 5,772 nucleotides (nt), with six AUG-initiated open reading frames, one non-AUG-initiated open reading frame, and three untranslated regions, showing typical features of the family Luteoviridae. Sequence comparison and phylogenetic analyses suggested that WLYaV had the closest relationship with sugarcane yellow leaf virus (ScYLV), but the identities of full genomic nucleotides and deduced amino acid sequence of coat protein (CP) were 64.9 and 86.2%, respectively, below the species demarcation thresholds (90%) in the family Luteoviridae. Furthermore, agroinoculation of Nicotiana benthamiana leaves with a cDNA clone of WLYaV caused yellowing symptoms on the plant. Our study adds a new polerovirus that is associated with wheat leaf yellowing disease, which would help to identify and control pathogens of wheat.

Keywords: Luteoviridae; Polerovirus; deep sequencing; infectious cDNA clone; leaf yellowing; wheat.

Figure 1

Symptoms on wheat samples collected from Jinan, China. (A) Symptomless, apparently healthy sample. (B,C) Sample JN-U3 infected with virus newly discovered, with dull yellowing and little dwarfing.

Figure 2

Length distribution of total sRNAs in sample JN-U3.

Figure 3

Analysis of WLYaV genome. (A) Characteristics of WLYaV genome, full length: 5,772 nt; 5′UTR: nt 1~58, with ACUAAA start; ORF0: nt 59~877, encodes a 31.4 kD protein (P0), a putative RNA-silencing suppressor (RSS); ORF1: nt 228~2,099, encodes a 69.1 kD putative polyprotein (P1); ORF2: nt 1,658~3,364, encodes a putative fusion protein of RdRp (P1–P2 fusion) by a −1 ribosomal frameshift with ORF1; ORF3a: nt 3,435~3,569, encodes a protein that may be involved in long-distance movement; ORF3: nt 3,553~4,143, encodes a 21.7 kD protein, a putative coat protein (P3); ORF4: nt 3,584~4,036, within ORF3, encodes a 17.0 kD protein, a putative movement protein (P4); ORF5: nt 4,144~5,577, encodes a putative fusion protein, a read-through protein (P3–P5 fusion) with ORF3 by suppression of termination; 3′UTR: nt 5,578~5,772, without a polyA tail. (B) Contigs mapped to the WLYaV genome: 11 of 8,798 contigs were remapped to the WLYaV genome, which covered 99% of the genome; the four numbered contigs were positioned using a blast search of known viruses. (C) Hotspots along the WLYaV genome, blue: positive strand, red: reverse strand. 5.25% of total sRNAs reads were derived from WLYaV, which were well-scattered on the viral positive and reverse strands except for several peaks at ORF0 (RSS), ORF3 (CP), and ORF5 (RTD).

Figure 4

Characteristics of the vsiRNAs. (A) Size distribution of vsiRNAs, 18~25 nt long, with 22- and 21-nt sRNAs predominated. (B) Percentage of A/U/G/C bases in vsiRNAs, a bias toward bases A and U especially in the anti-genome was found (52.06% in genome, 67.32% in anti-genome). Blue: genome, red: anti-genome.

Figure 5

Phylogenetic analyses of WLYaV with other viruses in the family Luteoviridae. The phylogenetic trees were generated using the neighbor-joining method by MEGA 6 software. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches. Phylogenetic tree based on (A) complete genome nucleotide sequences, deduced (B) RdRp and (C) CP amino acid sequences. The three phylogenetic trees had similar topologies, and both phylogenetic trees showed that WLYaV was most closely related to ScYLV and belonged to genus Polerovirus. Virus accession numbers are listed in Table S2.

Figure 6

Agroinoculation infectivity assay of cDNA clone on N. benthamiana. (A) Construction of infectious cDNA clone. Whole viral genome was cloned using fragments A and B digested with StuI, NcoI and SalI and ligated with StuI-SalI digested pCB301 vector, 2 × 35S: promoter, RZ: ribozyme, NOS: teminator. (B) Confirmation of the constructs obtained by PCR, M5: DL 5 000 DNA marker, 1: pEASYT5-A, 2: pEASYT5-B, 3: pCB301-WLYaV. (C) Symptoms on infected N. benthamiana 4 weeks after infiltration of lower 3~4 leaves; upper leaves are systemically infected. Left: inoculated with pCB301 as negative control, no obvious symptoms; right: inoculated with pCB301-WLYaV, stunted plants have leaf yellowing. (D) Detection of WLYaV in N. benthamiana by RT-PCR 14 days post-inoculation, M2: DL 2 000 DNA marker, 17/24 plants were positive, NC: negative control (pCB301-inoculated N. benthamiana), PC: positive control (JN-U3), +: exhibiting yellowing symptom.