Small RNA Profiles from Virus-Infected Fresh Market Vegetables

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Small RNA profiles from virus-infected fresh market vegetables Alessandra Frizzi, Yuanji Zhang, John Kao, Charles Hagen, and Shihshieh Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf503756v • Publication Date (Web): 11 Nov 2014 Downloaded from http://pubs.acs.org on November 16, 2014

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Journal of Agricultural and Food Chemistry

Small RNA profiles from virus-infected fresh market vegetables

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Alessandra Frizzi1,#, Yuanji Zhang2,#, John Kao3, Charles Hagen1 and Shihshieh Huang1,* 1

Calgene Campus, Monsanto Company, 1920 Fifth St, Davis, CA 95616, USA Chesterfield Campus, Monsanto Company, 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA 3 Monsanto Vegetable Seeds - Woodland, 37437 State Hwy 16, Woodland, CA 95695, USA 2

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These authors contributed equally to this work *Correspondence (fax +1 530 792-2005; email [email protected])

Keywords: Vegetable, small RNA, siRNA, miRNA, RNAi, RNA silencing, genetic

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engineering, Tomato spotted wilt virus (TSWV), Potato virus Y (PVY), Watermelon

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mosaic virus (WMV), Iris yellow spot virus (IYSV)

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ACS Paragon Plus Environment


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Abstract

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Functional small RNAs, such as short interfering RNAs (siRNAs) and microRNAs

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(miRNAs), exist in freshly consumed fruits and vegetables. These siRNAs can be

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derived from either endogenous sequences or from viruses that infect them.

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Symptomatic tomatoes, watermelons, zucchini and onions were purchased from

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grocery stores and investigated by small RNA sequencing. By aligning the obtained

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small RNA sequences to sequences of known viruses, four different viruses were

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identified as infecting these fruits and vegetables. Many of these virally-derived small

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RNAs along with endogenous small RNAs were found to be highly complementary to

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human genes. However, the established history of safe consumption of these

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vegetables suggests that this sequence homology has little biological relevance. By

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extension, these results provide evidence for the safe use by humans and animals of

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genetically engineered crops using RNA-based suppression technologies, especially

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vegetable crops with virus resistance conferred by expression of siRNAs or miRNAs

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derived from viral sequences.

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INTRODUCTION

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RNA interference (RNAi) or RNA silencing is a broadly used gene regulation

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mechanism in eukaryotes. It has been implicated in many aspects of biological

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processes such as regulation of growth and development, defense against pathogens

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and response to stress (1-3). Active research over the last decade has revealed the

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molecular basis of RNAi to a great extent (4-7). Most of the known RNAi components,

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some conserved across the kingdoms and some unique among few species, can be 2


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divided into two groups. They are either involved in generation of the small RNAs, or

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part of various RNAi complexes directly or indirectly associated with the small RNAs. At

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the core of the RNAi mechanism lies the small RNA whose primary sequence is used to

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guide the RNAi machinery to its specific gene target. The DNA or mRNA of the targeted

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genes, which bear a complementary sequence to the small RNA, is then modified by

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the RNAi machinery. These modifications, including DNA methylation, mRNA

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degradation or translational inhibition, eventually lead to the down-regulation of target

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genes and result in biological changes. Furthermore, these studies also found that

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exogenous small RNAs, whether they are either synthesized chemically or generated

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through transgenic expression can trigger RNAi with a similar biological effect as long

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as the required sequence signature is present.

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The characteristic of eukaryotic RNAi whereby the sequence signature of an

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exogenous small RNA can trigger a gene specific down-regulation in living organisms

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has immense implications in medicine and plant biotechnology. RNAi-based drugs

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treating diseases such as macular degeneration (AMD), cancer, asthma and glaucoma,

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or combating viruses including Respiratory syncytial virus (RSV), Hepatitis C virus

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(HCV) and Human immunodeficiency virus (HIV), are under clinical development (8, 9).

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In the case of human therapeutics, RNAi-based drugs have met with tremendous

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challenges presented by the need to achieve delivery in the face of a myriad of

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biological and physicochemical barriers (10, 11). These barriers have limited the

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efficacy, and thus the commercial success of this promising technology in therapeutics

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to date (9, 11). However, RNA-based suppression technology has been successfully

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applied in agriculture for more than a decade. The first transgenic food crop, Flavr Savr

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tomato, commercialized in 1994, employed antisense technology (now known as a part

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of the RNA silencing mechanism) to maintain fruit firmness for easy handling (12, 13).

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Subsequently, virus resistant squash and papaya were introduced to the market in the

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late 1990’s. Although it was once thought to be mediated by transgenically expressed

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viral coat protein, the virus resistance in these plants is more likely to result from

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transgenic siRNAs derived from the viral coat protein gene. A list of RNA silencing

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based biotech crops that are under development for commercial use was previously

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summarized by Frizzi and Huang (14).

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Although theoretically the 20+ nt size of small RNAs allow them to specifically

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identify their intended mRNA targets, mismatches and gaps in base-pairing between

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miRNAs or siRNAs and their target mRNAs are commonly found. This suggests that not

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all the bases of a small RNA are necessary for target recognition. In plants, mRNA

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slicing is the principal mode of post-transcriptional RNA silencing regulation; a guiding

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small RNA requires strong base-pairings with its target at nucleotides 2-13 but

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sequence identity is less critical at nucleotides 15-20 (15, 16). Because the RNAi

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mechanism effectively parses sequence mismatches and ambiguities, it has been

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shown through in vitro studies conducted with transfected small RNAs that small RNAs

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can cause gene suppression of unintended gene targets (17). This outcome, commonly

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referred to as the “off-target” effect has led to increased scrutiny regarding the

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specificity of RNAi-based drugs and also been proposed as a putative concern for food

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crops that are engineered to produce small RNAs. However, this “off-target” effect has

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been characterized using in vitro systems and is orders of magnitude less potent than

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“on-target” suppression (9, 11). Such suppression also requires special sequence

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contexts, thermodynamic criteria, target accessibility and other conditions (18, 19).

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To investigate the level of significance of regularly-consumed small RNAs that

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share sequence homology with human genes, the small RNA profiles of fresh market

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fruits and vegetables were examined. These were locally purchased tomatoes,

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watermelons, zucchini and onions that were selected because their appearance

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suggested they were likely to be infected with viral disease(s). Using a next-generation

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high throughput sequencing method, between 1.6 and 3.6 million small RNA sequences

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were obtained from each sample. Although most of the small RNAs found in these

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vegetables were likely derived from endogenous genome, up to 9.5% were identified as

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sequences originating from viruses. Many of these small RNAs were found to share

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sequence similarity with human genes. Small RNA profiles such as these resemble

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those of plants genetically modified to express fragments of virus sequences for the

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purposes of conferring virus resistance (14). The analysis of the small RNAs presented

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here provides insights into the plant-virus interaction and also evidence of a history of

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safe consumption for dietary small RNAs. By extension, this research supports the

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safety of biotechnology derived crops employing RNA-based gene suppression when

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consumed by humans or animals.

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MATERIALS AND METHODS

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Plant materials. Vegetables suspected of virus diseases based on physical

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characteristics were selectively purchased at local farmers’ markets or supermarkets in

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the region of Yolo County, California. The tomato fruits displayed lesions with a chlorotic

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concentric ring pattern typical of those caused by Tomato spotted wilt virus, (TSWV,

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family Bunyaviridae, genus tospovirus). The onion bulbs with attached green leaves

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exhibited elongated lesions typical of those caused by the Iris yellow spot virus (INSV,

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family Bunyaviridae, genus tospovirus). The squash fruits had mosaic symptoms

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consistent with those caused by viruses such as the Watermelon mosaic virus (WMV),

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Zucchini yellow mosaic virus (ZYMV) or Papaya ring spot virus (PRSV) (all viruses in

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the family Potyviridae, genus potyvirus). The melon fruit was chosen based on mottling

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symptoms that might be caused by a number of different potyviruses.

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Plant RNA isolation and small RNA sequencing. Symptomatic portions of the

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fruits/bulbs were harvested and ground in liquid nitrogen. Total RNA was extracted

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using TRIzol® (Invitrogen, Carlsbad, CA) following the manufacturer’s recommended

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protocol. RNA was quantified by Nanodrop® (Thermo scientific, Wilmington, DE), and

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its integrity was verified by gel electrophoresis. Small RNA library construction and

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sequencing was performed with Illumina technology (Illumina Inc, San Diego, CA) as

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described previously (20). Four to six million raw reads were generated from each

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library. After the 5’ and 3’ adaptors were identified and removed from the raw reads,

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reads with sequence length from 18 to 26 nt were parsed out for further analysis (Figure

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1). The sequences of these raw reads can be found in NCBI under BioProject accession

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PRJNA265505.

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Small RNA sequence analysis. The parsed small RNAs were mapped to plant

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virus sequences downloaded from www.dpvweb.net on 03/02/2012. Small RNA

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matching was performed using “SHRiMP” v2 (21). Only perfectly mapped small RNAs

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were counted. RNAi plays an important anti-viral role in plants. Upon virus infection,

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small RNAs are generated with the help of host plant Dicer-like protein from viral

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dsRNAs either as viral replicative intermediates or resulted from host RNA-dependent

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RNA polymerases on viral templates. The viral siRNAs are from both strands of the

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dsRNA, and predominantly 21 and 22 nt in length. To remove noise from the virus

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mapping results such as non-virus sequence contamination, we applied three filters: 1)

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A virus is called to have infested the plant if only its sequence has at least ten perfectly

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matched and sequence distinct small RNAs, 2) 21 and 22 nt small RNAs account for at

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least 60% of the small RNAs that mapped to a virus sequence, and 3) the abundance

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of small RNAs mapping to either strand of a virus sequences reflects a minimum of 5%

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of overall mapped small RNA abundance to the virus sequence. Virus-mapped small

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RNAs were then compared to the human reference RNA set downloaded from

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genome.ucsc.edu on 02/07/2012. For this analysis, up to two mismatches in 21 nt small

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RNAs and three mismatches in 22 nt small RNAs were allowed. Similarly, all small

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RNAs not mapped to virus sequences (‘non-virus-mapped’), which were presumably

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derived from endogenous plant sequences were also compared to the human reference

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RNA set. To match this subset of plant small RNAs against human reference genes,

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human miRNAs, ribosomal RNAs, small nucleolar RNAs and small nuclear RNAs were

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excluded from the reference RNA set and up to two mismatches were allowed for each

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match. Detailed lists of the virus-mapped small RNAs and small RNAs homologous to

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human genes can be found in the supplemental tables.

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RESULTS

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Deep sequencing of vegetable small RNA populations. RNA extracted from

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tomato (fruit), melon (fruit), zucchini (fruit) and onion (bulb and stem) was sequenced

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using Illumina technology. Small RNA sequences ranging in length from 18 to 26 nt

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were parsed out for subsequent analyses (Figure 1 and Table 1). The peaks shown

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here are typical of plant small RNA distribution, showing major peaks at 21 and 24 nt,

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consistent with plant dicer-like protein (DCL) cleavage products (22). The 21 nt class

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was more predominant than the 24 nt for all the samples but melon. The total number of

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reads varied from 1,636,524 to 3,609,317, with unique reads well represented in all the

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libraries (Table 1).

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Size class distribution of virus-mapped small RNAs. Several filters were

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applied to map the parsed plant-derived small RNA sequences to the virus genome

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collections obtained from www.dpvweb.net (see Small RNA sequence analysis in

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Materials and Methods). The 21-22 nt size class siRNAs are significantly more

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abundant than the 24 nt in all four samples (Figure 2), consistent with the size of small

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RNA products of post transcriptional gene silencing usually associated with viral

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infection. However, the number of reads of the virus-mapped melon small RNA

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population is over 100 fold less than in the other vegetable libraries (Table 1).

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Validation of virus-mapped matches. The genomic sequences of the top two

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virus matches generated from mapping the siRNA libraries to the virus sequence

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collection (except for the melon library where only one matching virus was identified)

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were plotted against the sequences of their respective small RNA matches to illustrate

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their distribution (Figure 3). In addition to applying the filters described earlier, the

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presence of small RNAs across the entire length of the putative virus hit serves as a

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validation criterion suggesting the legitimate presence of the virus in the sample (Figure

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3A, 3B, 3C, 3D and 3F). In the case of the melon sample, although the number of small

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RNA matches is smaller in number (possibly reflecting low virus titer in the tissue) the

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virus hit seems to be authentic. Conversely, a sparsely distributed match pattern

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(Figure 3E and 3G) is an indication that the matching small RNAs likely originated from

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sources other than the matched virus. Due to sequence similarities between viruses,

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some small RNAs match to one virus and also cross-match to another: for example, in

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the zucchini sample, most of the small RNAs mapped to Soybean mosaic virus, SMV

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(Figure 3E) were also mapped to WMV (Figure 3D) and in the onion sample, all the

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small RNAs mapped to Tomato yellow fruit ring virus, TYFRV (Figure 3G) also mapped

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to INSV (Figure 3F).

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In the case of the Tomato spotted wilt virus, segment S matched in the tomato

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sample (Figure 3A), the small RNA reads in both the sense and antisense orientations

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come predominantly from the regions corresponding to the non structural and

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nucleocapsid protein coding sequences, and noticeably less from the intergenic region

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that is not actively transcribed. For PVY (Figure 3B), WMV (Figure 3D), and INSV

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segment L (Figure 3F) all regions are evenly represented (notice the change of scale),

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consistent with the generation of a single transcript over the genomic segment. The

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relative abundant of these virus-derived small RNAs across the virus genome and their

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sequence signatures could also reveal the biochemical properties of plant small RNA

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processing. For example, using virus-derived small RNA sequences from TSWV

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infected tomato and Nicotiana benthamiana, details of small RNA differential processing

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were examined previously (23). Our small RNA sequencing results which include three

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additional viruses could be applied to such studies in the future.

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Characterization of tomato library reads. The tomato small RNA library was

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further investigated and mapped to the Monsanto in-house assembled tomato reference

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genome. Only sequences with perfect matches to the tomato and/or viral genomes are

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included in the analysis and these matches accounted for 78% of all reads (Table 3).

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The remaining, non-matching reads could be mostly sequencing errors and some

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representing single nucleotide polymorphisms (SNPs) since the sample is sourced from

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a different tomato variety than the reference sequence. As previously observed, the

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abundance of 21-22 nt classes appears to have been enhanced by the virus derived

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small RNAs (Figure 4A). The 24-nt class small RNAs, mostly derived from the

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endogenous tomato genome, are both abundant and rich in unique sequences (Figure

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4A and 4B). This is probably due to their roles in genome-wide transcriptional regulation

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and chromatin modification (22).

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Comparison of vegetable small RNAs to the human transcriptome. To

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evaluate if small RNAs derived from food have sequence similarity to human genes, we

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compared our libraries of small RNA sequences to small RNAs using a database of

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human reference RNAs. A recent study suggested that ingestion of a rice-derived

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miRNA matching a human gene over its open reading frame could cause transcript

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suppression in humans (24), claims which have been strongly challenged in the peer-

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reviewed literature by several independent research groups collectively (25-27).

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Nevertheless, similar matching criteria were used in this evaluation.

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We divided the small RNAs in each library into virus-mapped and non-virus-

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mapped reads, representing exogenous and endogenous sources for the sequences,

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respectively. To simplify this bioinfomatic evaluation, the positions of the mismatches,

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which sometimes can have various degree of impact in target gene suppression efficacy

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(15, 16), were not given special considerations. The results in Table 4 summarize that,

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when allowing up to 2 mismatches in the 21 nt size class and 3 mismatches in 22 size

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class, many human genes are homologous to virally-derived small RNAs. A total of

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1580, 43, 884 and 1399 human genes have at least one sub-region sharing sequence

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similarity to virus-mapped small RNAs in the tomato, melon, zucchini and onion small

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RNA libraries, respectively (Table S1, S2, S3 and S4). Except for the melon library,

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where fewer virus-mapped small RNAs were found, virus-derived small RNAs that

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match to human gene transcripts appear abundant. For example, in the tomato library,

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21816 reads or 11.1% of the virus-mapped reads are highly homologous to 1580

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human genes (Table 4). In some instances, thousands of virus-derived small RNAs can

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match a single human gene. The top three human genes matched by the most virus-

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mapped small RNAs in the tomato, zucchini and onion are listed in Table 5. However,

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there is no human gene match shared by the virus-derived small RNAs in all four

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libraries.

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Beyond the virus-derived small RNAs, the remaining small RNAs in these fruit or

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vegetable small RNA libraries are probably derived from endogenous plant sequences.

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These small RNAs can also share sequence homology to human genes. To eliminate

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trivial and low-complexity matches, the miRNAs, ribosomal RNAs, small nucleolar RNAs

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and small nuclear RNAs in the human reference RNA set that could be matched from

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the corresponding fruit or vegetable ribosomal RNA sequences were excluded from the

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sequence search. Up to two mismatches were allowed for each small RNA and the

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results are shown in Table 6. These results imply that up to 10.9% of the total small

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RNAs in regularly consumed vegetables share high degree of sequence similarity with

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human genes. Furthermore, 2774 human gene matches are shared by all four libraries

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(Table S5, S6, S7 and S8). This suggests that some human genes can have frequent

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matches by small RNAs derived from different fruits and vegetables. Although most of

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these matched sequences are few in numbers, numbers of endogenous small RNA with

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high copy numbers appear to have significant sequence similarity with human genes.

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For example, miRNA159a that is abundant in all four libraries can be mapped to a

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human gene encoding polycystic kidney and hepatic disease 1, PKHD1 protein (Table

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S5, S6, S7 and S8).

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DISCUSSION

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A typical plant small RNA size distribution is characterized by abundant 21 and

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24 nt size classes; the 24 nt class is commonly present at an abundance similar to, or

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more than that of the 21 nt size class (22). The 24 nt class small RNA is responsible for

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transcriptional regulation and chromatin modification (28), while the 21 nt class small

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RNA is mostly involved in post-transcriptional (29) and antiviral defense (30, 31). Except

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for the melon sample, the small RNA size profiles of all other fruit and vegetable

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samples displayed a higher abundance of 21 nt-sized small RNAs (Figure 1). This is

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likely due to the presence of viruses in these tissues that induced an antiviral RNA

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silencing response, resulting in the accumulation of 21 nt small RNAs. Indeed,

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significant amounts of total small RNA populations are found to have originated from

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viral sequences, which partially contributed to the greater abundance of the 21 nt class

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seen in tomato, zucchini and onion samples (Figure 2). However, directly comparing

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the small RNA populations between known infected and uninfected plants would be

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necessary to confirm these results.

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Previously, we have shown that it is possible to reassemble significant portions of

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TSWV genome from overlapping small RNA sequences obtained by small RNA

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sequencing of tomato infected with the virus (20). Here we report that several viruses

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can be easily and precisely detected in fresh market vegetables using small RNA

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sequencing. Furthermore, except for the WMV in the melon sample where the virus

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accumulation was likely to be low, the contigs assembled from the small RNA

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sequences from other samples embody large portions of the virus genomes. For

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example, as much as 89% of TSWV, 84% of PVY, 76% of WMV and 96% of INSV total

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genomic sequences are covered by the small RNAs in their respective samples (data

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not shown). Therefore, it is possible to use small RNA sequencing to assemble the

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genome of a novel virus especially when the endogenous small RNAs of host plant can

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be identified and excluded. With gradual reduction in cost since its invention, small RNA

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sequencing has the potential to be used as a one-step diagnostic and discovery tool for

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plant viruses in the future.

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Since RNA-mediated gene suppression in plants and animals operate through

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fundamentally similar mechanisms and synthetic dsRNA or siRNA/miRNA are known to

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work in plant or animal cells in vitro in a sequence-specific manner, some have

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cautioned the use of this RNA-based gene suppression in plant biotechnology.

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Recently, a study claiming that a miRNA, miR168a, from rice can target a mouse gene

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for suppression through oral ingestion has intensified the debate (24). The experimental

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design and assumptions of this study have been disputed (11, 27, 32). This

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phenomenon of significant miRNA uptake in mice, nonhuman primates and humans has

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not been reproduced by others (25-27) and the reported physiological impacts by Zhang

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and colleagues were not reproduced in a properly controlled feeding study with higher

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miR168a exposures (27). In addition, these observations are inconsistent with the

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weight of the evidence for the safety of ingested nucleic acids, as evidenced by

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biological barriers to their uptake and/or activity in higher organisms, their extensive

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history of safe consumption and the delivery challenges faced by pharmaceutical

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companies developing oligonucleotide based therapeutics (11, 33). The human diet is

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made up of largely eukaryotic sources which contain an abundance of small RNAs.

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Therefore, it is not surprising that such small RNAs were found, intact, in food through

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small RNA sequencing (24-27, 34). Studies have also demonstrated that some

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endogenous long dsRNAs in crop plants share sequence complementarity of at least 21

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nucleotides with human genes (33, 34).

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In the current study, we have sequenced the small RNAs in fruits and vegetables

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readily available for purchase, and likely to be ingested by consumers in an

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unprocessed or raw state. Sequences highly homologous to human genes were

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abundant in these fruits and vegetables, and we found that small RNAs originating

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within the plant and from infecting viruses can have matches to human genes. However,

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the consumption of fresh fruits and vegetables like these is not associated with an

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obvious heath risk - indeed the opposite is true (35, 36).

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It is logical to assume that small RNAs produced by transgenes are

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fundamentally no different than endogenously-derived plant small RNAs. The data

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presented here provide additional support for the assumption that small RNAs derived

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from transgenic sources are no more impactful to human health than the multitudes of

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endogenous plant small RNAs present in foods regularly consumed every day. This

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opinion is shared by the independent statutory agency Food Standards Australia New

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Zealand (FSANZ) who have noted that “there is no scientific basis for suggesting that

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small dsRNAs present in some GM foods have different properties or pose a greater

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risk than those already naturally abundant in conventional foods” (37). Coupled with the

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long history of safe consumption of these dietary small RNAs, the results described

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here provide further evidence to suggest that RNA-based gene suppression in biotech

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crops is unlikely to present a direct safety hazard to humans or animals.

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ABBREVIATIONS USED

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TSWV, Tomato spotted wilt virus; PVY, Potato virus Y; WMV, Watermelon mosaic virus;

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SMV, Soybean mosaic virus; INSV, Iris yellow spot virus, TYFRV, Tomato yellow fruit

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ring virus; ZYMV, Zucchini yellow mosaic virus; PRSV, Papaya ring spot virus.

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ACKNOWLEDGEMENTS

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We thank Daniel Ader, Mingya Huang and Mitchell Sudkamp for constructing the small

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RNA libraries and performing the small RNA sequencing, and Jay Petrick for his

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valuable comments on the manuscript. We also thank Sofia Castiglioni for her

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contribution to the artwork in the Table of Contents/Abstract Graphic.

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17



420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465

Figure/Table legends Figure 1. Relative abundance of small RNA size classes. The relative abundance of small RNA with length ranging from 18-26 nt is shown in the graph. All the samples had the typical plant small RNA size distribution with peaks at 21 and 24 nt. With the exception of the melon sample, the 21 nt class was more abundant than the 24 nt class. Figure 2. Relative abundance of small RNA size classes from small RNAs mapped to the virus sequence database. For all samples, most of the virus-mapped reads belong to the 21 and 22 nt size classes. These small RNAs, usually associated with posttranscriptional gene silencing, are likely generated upon viral infection. Figure 3. Validation of virus-mapped small RNA reads to their respective matching viral sequences. The top two virus matches for each sample were plotted against small RNAs mapped to them. Except for E and G, the small RNAs are mapped across the entire virus sequences. Therefore, there is a high probability that such virus is present in the samples. On the contrary, only sparse matches were found on E and G, which suggests that these mapped small RNAs were derived from different sources. Figure 4. Categories of reads and unique reads in small RNA size classes from the tomato sample. The total number of reads (A) and the unique number of reads (B) of tomato small RNAs that matched perfectly to the in-house assembled reference tomato genome (red line), virus genome database (green line) or both (purple line). Together they account for 78% (Table 3) of the all reads (blue line). Table 1. Total numbers of small RNA reads obtained from each sample library and the numbers of reads that mapped to the virus sequences. Only parsed (18-26 nt) reads were used in the mapping. Table 2. The top two virus matches and the numbers of small RNA reads matched in each sample library. The melon sample had only one virus match. For viruses that have more than one genome segments, only one segment is shown in the table, though all other segments had similar significant matches. Table 3. The number of total and unique small RNA reads in each category in the tomato sample. Table 4. Virus-mapped small RNA reads sharing sequence homology to human reference RNA set. The virus-mapped reads, 21- and 22-nt size classes, in each sample (Figure 2) were matched against the human reference RNA set downloaded from genome.ucsc.edu (02/07/2012), allowing up to two or three mismatches for each 21- and 22-nt small RNA read, respectively. Table 5. Examples of human RNAs matching the sequence of virus-mapped small RNA reads. Except for the melon sample where only few matches were found, the top three human RNA matches in each sample are listed. The majority of the reads matched to a 18


Page 18 of 31

Page 19 of 31

466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504


single sub-region of the transcript as shown using the base number referenced in GenBank. Table 6. Non-virus-mapped small RNA reads sharing sequence homology to human reference RNA set. The virus-mapped small RNA reads were removed from each small RNA library. The rest of the small RNA reads, presumably derived from endogenous sequences, were matched against the human reference RNA set downloaded from genome.ucsc.edu. The miRNAs, ribosomal RNAs, small nucleolar RNAs and small nuclear RNAs were excluded from the human reference RNA set and allowed up to two mismatches for each plant small RNA. The number of human genes matched as well as the numbers of unique and total small RNA reads whose sequences matched these human genes are shown. Supplemental tables (all in an Excel file) Table S1. List of virus-mapped small RNA reads in the tomato sample that share sequence homology to human reference RNA set. Table S2. List of virus-mapped small RNA reads in the melon sample that share sequence homology to human reference RNA set. Table S3. List of virus-mapped small RNA reads in the zucchini sample that share sequence homology to human reference RNA set. Table S4. List of virus-mapped small RNA reads in the onion sample that share sequence homology to human reference RNA set. Table S5. List of non-virus-mapped small RNA reads in the tomato sample that share sequence homology to human reference RNA set. Table S6. List of non-virus-mapped small RNA reads in the melon sample that share sequence homology to human reference RNA set. Table S7. List of non-virus-mapped small RNA reads in the zucchini sample that share sequence homology to human reference RNA set. Table S8. List of non-virus-mapped small RNA reads in the onion sample that share sequence homology to human reference RNA set.

19



Page 20 of 31

Small RNA profiles from virus-infected fresh market vegetables, Frizzi et al. – Table of Contents/Abstract Graphics

Plant virus

Small RNAs

Endogenous

Virus derived

? Human mRNAs

1 ACS Paragon Plus Environment

Page 21 of 31


Small RNA profiles from virus-infected fresh market vegetables, Frizzi et al. – Figure 1

Relative abundance

50%

Tomato Melon

40%

Zucchini Onion

30% 20% 10% 0% 18

19

20

21

22

23

24

25

26

Length (nt) Figure 1. Relative abundance of small RNA size classes. The relative abundance of small RNA with length ranging from 18-26 nt is shown in the graph. All the samples had the typical plant small RNA size distribution with peaks at 21 and 24 nt. With the exception of the melon sample, the 21 nt class was more abundant than the 24 nt class.



Page 22 of 31


Relative abundance

60%

Tomato

50%

Melon Zucchini

40%

Onion 30% 20% 10% 0% 18

19

20

21

22

23

24

25

26

Length (nt) Figure 2. Relative abundance of small RNA size classes from small RNAs mapped to the virus sequence database. For all samples, most of the virus-mapped reads belong to the 21 and 22 nt size classes. These small RNAs, usually associated with post-transcriptional gene silencing, are likely generated upon viral infection.


1 316 631 946 1261 1576 1891 2206 2521 2836 3151 3466 3781 4096 4411 4726 5041 5356 5671 5986 6301 6616 6931 7246 7561 7876 8191 8506 8821 9136 9451 9766

Read abundance (blue: positive strand; red: negative strand)

0 0

-2000 - 50

-4000 - 100

-6000 - 150

-8000 - 200 1 314 627 940 1253 1566 1879 2192 2505 2818 3131 3444 3757 4070 4383 4696 5009 5322 5635 5948 6261 6574 6887 7200 7513 7826 8139 8452 8765 9078 9391

1 97 193 289 385 481 577 673 769 865 961 1057 1153 1249 1345 1441 1537 1633 1729 1825 1921 2017 2113 2209 2305 2401 2497 2593 2689 2785 2881

Page 23 of 31 Journal of Agricultural and Food Chemistry


A Tomato/Tomato spotted wilt virus, segment S B Tomato/Potato virus Y

8000 200

6000 150

4000 100

2000 50

C Melon/Watermelon mosaic virus

4

3

2

1

-1 0

-2

-3

-4

4



Page 24 of 31

Small RNA profiles from virus-infected fresh market vegetables, Frizzi et al. – Figure 3 continued

1 326 651 976 1301 1626 1951 2276 2601 2926 3251 3576 3901 4226 4551 4876 5201 5526 5851 6176 6501 6826 7151 7476 7801 8126 8451 8776 9101 9426 9751

F Onion/Iris yellow spot virus, segment L

400 300 200 100 0 -100 -200 -300 -400 -500 -600

G Onion/Tomato yellow fruit ring virus, partial L gene

4000

400

3000

300

2000

200

1000

100

0

0

-1000

1 - 00

-3000

2 - 00

1 288 575 862 1149 1436 1723 2010 2297 2584 2871 3158 3445 3732 4019 4306 4593 4880 5167 5454 5741 6028 6315 6602 6889 7176 7463 7750 8037 8324 8611

-2000

1 28 55 82 109 136 163 190 217 244 271 298 325 352 379 406 433 460 487 514 541 568 595 622 649 676 703 730 757 784 811

3000 2000 1000 0 -1000 -2000 -3000 -4000 -5000 -6000 -7000

1 311 621 931 1241 1551 1861 2171 2481 2791 3101 3411 3721 4031 4341 4651 4961 5271 5581 5891 6201 6511 6821 7131 7441 7751 8061 8371 8681 8991 9301

E Zucchini/Soybean mosaic virus

D Zucchini/Watermelon mosaic virus

Genome/gene base coverage Figure 3. Validation of virus-mapped small RNA reads to their respective matching viral sequences. The top two virus matches for each sample were plotted against small RNAs mapped to them. Except for E and G, the small RNAs are mapped across the entire virus sequences. Therefore, there is a high probability that such virus is present in the samples. On the contrary, only sparse matches were found on E and G, which suggests that these mapped small RNAs were derived from different sources. 5 ACS Paragon Plus Environment

Page 25 of 31



B

A 1048576 262144 65536 16384 4096 1024 256 64 16 4 1

Number of reads

Number of unique reads

262144

18 19 20 21 22 23 24 25 26

65536 16384 4096 1024 256 64 16 4 1 18 19 20 21 22 23 24 25 26

Length (nt)

Length (nt)

All Tomato genome-mapped Virus-mapped Overlap

Figure 4. Categories of reads and unique reads in small RNA size classes from the tomato sample. The total number of reads (A) and the unique number of reads (B) of tomato small RNAs that matched perfectly to the inhouse assembled reference tomato genome (red line), virus genome database (green line) or both (purple line). Together they account for 78% (Table 3) of the all reads (blue line). 6 ACS Paragon Plus Environment


Page 26 of 31

Small RNA profiles from virus-infected fresh market vegetables, Frizzi et al. – Table 1

Parsed reads (18-26 nt)

Solanum lycopersicum Tomato RNA (fruit)

Virus-mapped reads Unique Total reads Unique reads Total reads reads 5315393 3609317 1209206 196069 23929

Melon

Cucumis melo

Melon RNA (fruit)

4825754

1636524

536672

185

175

Zucchini

Cucurbita pepo

Zucchini RNA (fruit)

5184745

3344051

838271

191085

22949

Allium cepa

Onion RNA (bulb + stem) 6038349

3145549

915603

301913

33144

Sample ID Tomato

Onion

Species

Tissue

Raw reads

Table 1. Total numbers of small RNA reads obtained from each sample library and the numbers of reads that mapped to the virus sequences. Only parsed (18-26 nt) reads were used in the mapping.


Page 27 of 31



Sample Tomato Melon Zucchini Onion

Virus species matched

Mapped reads

GenBank accession

Tomato spotted wilt virus (TSWV), segment S

HQ402595

Total reads 100079

Potato virus Y (PVY)

HQ912865

12076

4040

Watermelon mosaic virus (WMV)

FJ623474

128

122

Watermelon mosaic virus (WMV)

EU660583

146333

17542

D00507

3607

419

Iris yellow spot virus (INSV), segment L

FJ623474

108325

15979

Tomato yellow fruit ring virus (TYFRV), partial L gene

AJ493271

802

91

Soybean mosaic virus (SMV)

Unique reads 6211

Table 2. The top two virus matches and the numbers of small RNA reads matched in each sample library. The melon sample had only one virus match. For viruses that have more than one genome segments, only one segment is shown in the table, though all other segments had similar significant matches.



Page 28 of 31


Sample Tomato

All reads Unique Total Reads Reads 3609317 120206

Tomato genome-mapped reads Total reads 2596723

Virus-mapped reads

Overlap reads

Unique reads Total Reads Unique Reads Total reads 824996 196069 23929 2374

Unique reads 676

Table 3. The number of total and unique small RNA reads in each category in the tomato sample.


Page 29 of 31



Sample

Number of human RNAs matched

Number of unique reads

Total reads

Tomato

1580

1786

21816

% of total virus-mapped reads sharing homology with human genes 11.1%

Melon

43

22

22

11.9%

Zucchini

884

1558

16634

8.7%

Onion

1399

1936

18005

6.0%

Table 4. Virus-mapped small RNA reads sharing sequence homology to human reference RNA set. The virusmapped reads, 21- and 22-nt size classes, in each sample (Figure 2) were matched against the human reference RNA set downloaded from genome.ucsc.edu (02/07/2012), allowing up to two or three mismatches for each 21- and 22-nt small RNA read, respectively.



Page 30 of 31


Sample

Human RNA matched

GenBank ID

Region matched

Multiple EGF-like-domains 8 (MEGF8), mRNA NM_001410 10253-10274 Family with sequence similarity 190, member A (FAM190A), NM_001145065 5152-5170 Tomato transcript variant 1, mRNA Rho-associated, coiled-coil containing protein kinase 2 NM_004850 4525-4545, 997-1017 (ROCK2), mRNA WD repeat domain 3 (WDR3), mRNA NM_006784 3424-3444 Homo sapiens regulator of G-protein signaling 7 (RGS7), NM_002924 1273-1295 Zucchini mRNA Proline-rich transmembrane protein 2 (PRRT2), transcript NM_145239 1444-1464 variant 1, mRNA Triosephosphate isomerase 1 pseudogene 3 (TPI1P3), nonNR_027338 639-659 coding RNA Olfactory receptor, family 6, subfamily P, member 1 (OR6P1), Onion NM_001160325 635-654 mRNA DYX1C1-CCPG1 readthrough (non-protein coding) (DYX1C12962-2983, 3037NR_037923 CCPG1), non-coding RNA 3056

Number of Total reads unique reads 20 3451 1

2419

6

2337

6

1019

14

904

4

462

6

1043

10

941

11

689

Table 5. Examples of human RNAs matching the sequence of virus-mapped small RNA reads. Except for the melon sample where only few matches were found, the top three human RNA matches in each sample are listed. The majority of the reads matched to a single sub-region of the transcript as shown using the base number referenced in GenBank.


Page 31 of 31



Sample Tomato Melon Zucchini Onion

Number of human RNAs Number of unique reads Total reads matched

% of total non-virus-mapped reads sharing homology with human genes

13683 8664

33189 14961

337610 157019

9.4% 9.6%

8286 13851

14633 34193

144654 342541

4.3% 10.9%

Table 6. Non-virus-mapped small RNA reads sharing sequence homology to human reference RNA set. The virusmapped small RNA reads were removed from each small RNA library. The rest of the small RNA reads, presumably derived from endogenous sequences, were matched against the human reference RNA set downloaded from genome.ucsc.edu. The miRNAs, ribosomal RNAs, small nucleolar RNAs and small nuclear RNAs were excluded from the human reference RNA set and allowed up to two mismatches for each plant small RNA. The number of human genes matched as well as the numbers of unique and total small RNA reads whose sequences matched these human genes are shown.


Small RNA Profiles from Virus-Infected Fresh Market Vegetables

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