Discovery and small RNA profile of Pecan mosaic-associated virus, a novel potyvirus of pecan trees

Abstract

A novel potyvirus was discovered in pecan (Carya illinoensis) showing leaf mosaic symptom through the use of deep sequencing of small RNAs. The complete genome of this virus was determined to comprise of 9,310 nucleotides (nt), and shared 24.0% to 58.9% nucleotide similarities with that of other Potyviridae viruses. The genome was deduced to encode a single open reading frame (polyprotein) on the plus strand. Phylogenetic analysis based on the whole genome sequence and coat protein amino acid sequence showed that this virus is most closely related to Lettuce mosaic virus. Using electron microscopy, the typical Potyvirus filamentous particles were identified in infected pecan leaves with mosaic symptoms. Our results clearly show that this virus is a new member of the genus Potyvirus in the family Potyviridae. The virus is tentatively named Pecan mosaic-associated virus (PMaV). Additionally, profiling of the PMaV-derived small RNA (PMaV-sRNA) showed that the most abundant PMaV-sRNAs were 21-nt in length. There are several hotspots for small RNA production along the PMaV genome; two 21-nt PMaV-sRNAs starting at 811 nt and 610 nt of the minus-strand genome were highly repeated.

Figure 1. The mosaic symptom of pecan leaves.

Figure 2. Genome organization of PMaV.

Dash areas represent 5′ UTR and 3′ UTR; the numbers above the figure represent the number of nucleotide of each proteins; the numbers below the figure represent the molecular mass of each proteins; the arrow represents protein PIPO in P3 protein zone, which is located at 2,511–2,750 nt of the PMaV genome; the 9 cleavage sites on this polypeptide also below the figure.

Figure 3

Neighbor-joining phylogenetic tree of Potyviridae viruses based on full genomic sequences (a) and CP amino acid sequences (b). Branch numbers represent the percent similarity in the bootstrap values out of 1000 replicates.

Figure 4. Filamentous virion in a leaf dip from infected pecan leaves with mosaic symptom.

The bar represents 200 nm.

Figure 5. Small RNA profiling of PMaV from infected pecan leaves.

(a) Bar graph showing the mapped PMaV-sRNA size distributions; (b) Relative frequency of 5′-terminal nucleotide for 20 to 24-nt PMaV-sRNAs; (c) Statistical analysis of PMaV-sRNAs mapped to the genomic or antigenomic sequences; (d) Genome-wide mapping of PMaV-sRNAs. The horizontal axis represents the relative position along the PMaV genome. The vertical axis represents the number of PMaV-sRNAs reads mapped to the PMaV genomic (+) or antigenomic (−) sequences. Arrows represent 811 nt and 610 nt of the minus-genomic RNA sequence.