The Sisal Virome: Uncovering the Viral Diversity of Agave Varieties Reveals New and Organ-Specific Viruses

Abstract

Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.

Keywords: Agave; metatranscriptomics; virome.

Figure 1

Strategy applied to uncover the virome of Agave species. The dot in the map indicates the localization of Monteiro, in the state of Paraíba. Agave fourcroydes, Agave sisalana and Agave Hybrid 11648 (and their respective organ samples) are indicated in different tones of green, for differentiation purposes. The following methods are indicated in the lower portion of the figure; RNA extraction, DNA extraction, high-throughput sequencing in the Illumina platform, and bioinformatics analysis (see further details in the Material and Methods section).

Figure 2

Overview of sequence similarity searches. The bar plots indicate the non-normalized values of viral, unknown, and other (non-viral) contigs in the nine analyzed samples. Color keys are indicated in the figure. Percentages are indicated inside color bars or on the right side, for viral contigs values. “Unknown” represents sequences with no significant hit at NCBI nucleotide and protein databases, while “non-viral” indicates sequences with hits with any other organism but viral.

Figure 3

Phylogenetic analysis of high-confidence viral sequences identified in Agave species. Phylogenetic trees contain 13 selected species, highlighted in blue. (A) Family Betaflexiviridae. In blue; Carlavirus. In green; Trinivirinae. In yellow; unclassified Betaflexivirus. (B) +ssRNA (positive sense, single-stranded RNA), in red. In orange; Ampelovirus. (C) Riboviria (in violet). Node support values were determined using 1000 pseudoreplicates where values over 60% of confidence are shown.

Figure 4

Principal component analysis of virus abundance in Agave organs. Viral species were used according to their abundance (green arrows) and presence in organs; Each organ (dots) is represented by a color: leaves (green dots), stems (yellow dots), and roots (brown dots).

Figure 5

Viral abundance of Agave-associated viruses by organ. Pie charts indicate viral family abundance by expression in tpm, in (A): leaves, (B): stems, (C): roots, and (D): in total. Values are summed for all plants. (E): The stacked bar plot indicates the relative abundance of the top 12 expressed viral species in each organ.

Figure 6

Organ tropism of Agave-infecting viruses. Colors indicate the presence (yellow) or absence (grey) of viral species over plant organs.