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