Identification of Three Viruses Infecting Mulberry Varieties

Abstract

Viruses-mediated genome editing in plants is a powerful strategy to develop plant cultivars with important and novel agricultural traits. Mulberry alba is an important economic tree species that has been cultivated in China for more than 5000 years. So far, only a few viruses have been identified from mulberry trees, and their application potential is largely unknown. Therefore, mining more virus resources from the mulberry tree can pave the way for the establishment of useful engineering tools. In this study, eight old mulberry plants were gathered in seven geographic areas for virome analysis. Based on transcriptome analysis, we discovered three viruses associated with mulberries: Citrus leaf blotch virus isolate mulberry alba 2 (CLBV-ML2), Mulberry-associated virga-like virus (MaVLV), and Mulberry-associated narna-like virus (MaNLV). The genome of CLBV-ML2 was completely sequenced and exhibited high homology with Citriviruses, considered to be members of the genus Citrivirus, while the genomes of MaVLV and MaNLV were nearly completed lacking the 5' and 3' termini sequences. We tentatively consider MaVLV to be members of the family Virgaviridae and MaNLV to be members of the genus Narnavirus based on the results of phylogenetic trees. The infection experiments showed that CLBV-ML2 could be detected in the inoculated seedlings of both N. benthamiana and Morus alba, while MaVLV could only be detected in N. benthamiana. All of the infected seedlings did not show obvious symptoms.

Keywords: Citrivirus; Narnavirus; Virgaviridae; mulberry alba; transcriptome; virus.

Figure 1

The heatmap shows the distribution of MaVLV, CLBV-ML2, and MaNLV in samples.

Figure 2

Predicted open reading frames (ORFs, with boxes), conserved motifs, domains, and viral proteins (different colors). (A) MaVLV, (B) CLBV-ML2, and (C) MaNTL. Viral methyltransferase (MT, pfam01660), viral helicase (HEL, pfam01443), and RNA-dependent RNA polymerase_2 (RdRp, pfam00978), 2OG-FeII Oxy superfamily (2OG-F, cl21496), peptidase C23 superfamily (Pep, cl05111), SP24 (pfam16504), coat protein (CP) and movement protein (MP). The unknown sequences of 5′ and 3′ UTRs are shown by black lines at both extremities, An: poly A tail.

Figure 3

Phylogenetic trees of (A) RdRp proteins constructed for representative members of the family Virgaviridae (40 tobamoviruses; 5 furoviruses; 6 pomoviruses; 2 pecluviruses; 3 tobraviruses and 32 unclassified virgaviridaes). (B) The pairwise identity plots of the RNA-dependent RNA polymerase amino acid sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red. 40 tobamovirus are collapsed together. Relationship: pink background.

Figure 3

Phylogenetic trees of (A) RdRp proteins constructed for representative members of the family Virgaviridae (40 tobamoviruses; 5 furoviruses; 6 pomoviruses; 2 pecluviruses; 3 tobraviruses and 32 unclassified virgaviridaes). (B) The pairwise identity plots of the RNA-dependent RNA polymerase amino acid sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red. 40 tobamovirus are collapsed together. Relationship: pink background.

Figure 4

Phylogenetic trees constructed for representative members of the Citrivirus genus. (A) The whole sequences for phylogenetic trees. (B) The pairwise identity plots of the complete genomic nucleotide sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red; relationship: pink background.

Figure 5

Phylogenetic trees of (A) RdRp proteins constructed for representative members of the family Narnaviridae (43 unclassified narnaviridaes; 32 unclassified narnaviruses; 2 narnaviruses). (B) The pairwise identity plots of the RNA-dependent RNA polymerase amino acid sequences aligned by ClustalW and displayed by sequence demarcation using TBtools software. The trees were constructed using the maximum likelihood method, and the statistical significance of the branches was evaluated by bootstrap analysis (1000 replicates). Newly discovered viruses are marked in red, unclassified narnaviridaes in black, unclassified narnaviruses in bule, and narnaviruses in green. Relationship: pink background.

Figure 6

The detection of RdRp protein and UTR sequences. (A) The expression of RdRp proteins of CLBV-ML2, MaVLV, and MaNLV by RT-PCR. (B) RACE amplification of CLBV-ML2. M: Marker. Red boxes: 5’UTR and 3’UTR target strips.

Figure 7

Symptoms on leaves of N. benthamiana and Morus alba infected by MaVLV, CLBV-ML2, and virus detection. (A) No symptoms on leaves of N. benthamiana and Morus alba. (B) RT-PCR detection of the infected leaves at 10 dpi. Lane 1 and 2: N. benthamiana, Lane 3: Morus alba, S: sample cDNA.

Figure 7

Symptoms on leaves of N. benthamiana and Morus alba infected by MaVLV, CLBV-ML2, and virus detection. (A) No symptoms on leaves of N. benthamiana and Morus alba. (B) RT-PCR detection of the infected leaves at 10 dpi. Lane 1 and 2: N. benthamiana, Lane 3: Morus alba, S: sample cDNA.