Detecting Viruses and Viroids in Tomato using Small RNA Deep Sequencing and VirusDetect Pipeline

Kai-Shu LingResearch Plant Pathologist

USDA-ARS, U. S. Vegetable LaboratoryCharleston, SC, USA

(AAFC-USDA, Cleveland, August 3, 2019)

World leading tomato producing countries(FAO statistics-2012, tonnes)

0

10000000

20000000

30000000

40000000

50000000

60000000

0

500,000

1,000,000

1,500,000

2,000,000

UnitedStates Canada

Mexico

Greenhouse tomato Fresh field tomato

North America Fresh Tomato Production (Mt)-2003

Controlled Environment Agriculture/Tomato Production

Arizona

A major viroid (TCDVd) outbreak in the U.S.

Seed-borne Viruses and Viroids on TomatoTobamovirus complex (4 species)

• Tobacco mosaic virus (TMV)• Tomato mosaic virus (ToMV)• Tomato mottle mosaic virus (ToMMV)• Tomato brown rugose fruit virus (ToBRFV)

Pospiviroid complex (8 species)• Potato spindle tuber viroid• Tomato chlorotic dwarf viroid• Tomato planta macho viroid (Mexican papita viroid)• Citrus exocortis viroid• Tomato apical stunt viroid• Columnea latent viroid• Pepper chat fruit viroid• Chrysanthemum stunt viroid

ToMV

MPVd

First report of ToBRFV in the U.S.

ToBRFV

PepMV

Worldwide emergence of ToBRFV• Middle East (2014-):

• Jordan: (Salem et al., 2016)• Israel: (Luria et al., 2017)• Palestine (Alkowni et al, 2019)• Turkey (Fidan et al., 2019)

• North America (2018-)• Mexico (Cambron-Crisantos et al.,

2019; Camacho-Beltran et al. 2019)• USA (Ling et al., 2019)• Canada (Ontario?)

• Europe (2018-): • Germany (Menzel et al., 2019)• Italy (Panno et al., 2019)• Belgium (?)

• Asia (2019-):• China (Yan et al., 2019)

Genetic diversity of Tobamoviruses

Nucleotide Substitution per 100 residues0

17.1

246810121416

KP861747 ToMMV 3801 IsraelKP861748 ToMMV 6742 IsraelHQ593616.ToMV-IranKP202857 ToMMV 10-100 FLKT810183 ToMMV NY-13JX121570 ToMV Iran2KT222999 ToMMV Capao Bonito BraAF411922 ToMV Sao PauloJX025564 ToMV China PepinoAJ243571 ToMV K1NC_028478_TBRFV_JordanFR878069_TMV Ohio V

ToMMV

ToMV

TMVToBRFV

Natural tomato infecting viroids in North America

Mexican papita viroidCanada, Mexico

Tomato chlorotic dwarf viroidCanada, Arizona, Mexico,

Potato spindle tuber viroid California, North Carolina

Nucleotide Substitution per 100 residues0

22.6

5101520

PSTVd_3077740PSTVd_NEPSTVd_CA1PSTVd_CapsicumPSTVd_NZPSTVd_NC12-01TCDVd_AF162131TCDVd_AZ1TCDVd_JapanTCDVd_UKTCDVd-Mexico_HM2TCDVd_AZ2MPVd_OG6-5MPVd-Canada_VF2MPVd_OG6-3MPVd_OG6-1MPVd_OG5MPVd_OG4MPVd_OG3MPVd_OG1MPVd_OG2MPVd-Mexico_Mex8TPMVd_K00817TASVd_IsraelTASVd_X06390TASVd_Sj1TASVd_SjTASVdTASVdCEVd-t Citrus exocortis viroid?

PSTVd

TCDVd

MPVd

TASVdTomato apical stunt viroid

CEVd

(From Agrios’ Plant Pathology, 3rd ed.)

Coat protein

RNA or DNA

Serological(ELISA)

Detection:

Virus structure, composition and strategies for detection

Molecular(PCR)

Common methods in plant virus detection

RNA or DNACoat protein

Indirect Tests (infectious + dead viruses) Direct Tests (infectious)

Tobamovirus

Next generation sequencing for plant virus detection

Host Virus RNA template NGS technology Reference

Tomato & Gomphrenaglobosa

PepMV, GMMV Total plant RNA Roche 454 Adams et al., 2009

Grapevine 4 RNA viruses + 3 viroids

Total plant RNA & dsRNA

Roche 454 Al Rwahnihet al. 2009

Grapevine GLRaV-3 & 3 others dsRNA Illumina Coetzee et al. 2010

Sweetpotato SPFMV, SPCSV and 2 DNA viruses

siRNA Illumina Kreuze et al., 2009

Tomato , Squash TSWV, SLCV siRNA Illumina Hagen et al., 2011

Grapevine HSVd, GYSVd siRNA Illumina Navarro et al., 2009

Viral siRNA biogenesis

AAARDR

A A A

AAA

AAA

siRNA (21-24 nt)

AAA

RISC

Gene silencing/Viral RNA degradation

Slicing

AGO AGO AGO AGO AGO

DCL

AGO

AGO

Dicing

Guiding

DCL

raw Illumina sRNA reads

cleaned sRNA reads

remove barcode & adaptor

align to tomato genome*

unmapped readstomato reads

contigsde novo assembly (velvet)

BLAST against nr and nt database

virus genome contigs

distribution of sRNA reads on virus genome contigs

align sRNA reads to virus genomes

tomato miRNAs

VirusDetect: Small RNA Bioinformatics Pipeline

B

C

VirusDetect: alignments of virus contigs to the reference virus genome

P1 CI NIa-VPg

NIa-ProP3

1 653 74 8 92 10 kb0

CPHC-Pro NIbAAA

Identification of a novel potyvirus (tomato necrotic stunt virus) from Mexico

siRNAContig

Validation and gap filling by RT-PCR and Sanger sequencing

Complete genome of an emerging CGMMV isolate in North America (Canada)

MET HEL RdRpMP

CP

(Li et al., 2015. Genome Announc 3(3):e00452-15)

Summary Deep sequencing of small RNA has revolutionized virus

detection and disease diagnosis for known or unknown viruses and viroids.

The VirusDetect pipeline provides an effective bioinformatics tool for data analysis and virus detection. It can also be applied for RNA-sequence data analysis.

This technology can be used to differentiate closely related strains in a mixed infection.

Validation and gap filling could be accomplished through PCR and Sanger sequencing.

Acknowledgments

USDA-ARS, Charleston-Chellappan Padmanabhan-Md Shamimuzzaman-Bidisha Chanda-Rugang Li-Andrea Gilliard

Funding:-USDA-NIFA SCRI Grants-USDA-ARS NPDRS program

Boyce Thompson Institute-Zhangjun Fei

Greenhouse tomato growers in North America