Construction of Full-Length Infectious Clones of Turnip Mosaic Virus Isolates Infecting Perilla Frutescens and Genetic Analysis of Recently Emerged Strains of Tumv in Korea

DOI: https://doi.org/10.21203/rs.3.rs-867651/v1

Abstract

Perilla is an annual herb with a unique aroma and taste and has been cultivated in Korea for hundreds of years. Owing to the highly edible and medicinal value of Perilla plants, it has been widely cultivated in many Asian and European countries. Recently, several viruses have been reported to cause diseases in Perilla in Korea, including turnip mosaic virus (TuMV) which is known as a brassica pathogen due to its significant damage to brassica crops. In this study, we determined the complete genome sequences of two new TuMV isolates originating from Perilla in Korea. Full-length infectious cDNA clones of these two isolates were constructed and their infectivity was tested by agroinfiltration on Nicotiana benthamiana and sap inoculation on Chinese cabbage and radish. In addition, we analyzed the phylogenetic relationship of six new Korean TuMV isolates and determined their respective affiliation with the four major groups. We also conducted recombination analysis for isolates recently occurring in Korean using RDP4 software, which provided new insight into the evolutionary relationships among Korean isolates of TuMV.

Introduction

Perilla (Perilla frutescens Britton), belonging to the family Lamiaceae that is composed of 236 genera and more than 7000 species, is an annual herb with a unique aroma and taste, and has been cultivated in Korea for hundreds of years [13]. Perilla is also cultivated in many other countries, such as China, Japan and Vietnam. Futhermore, because of its increasing economic significance, some western countries including Europe, the United States and Russia are growing Perilla [4]. Perilla is a very common and popular leafy vegetable in South Korea, which is usually eaten as a kind of salted vegetable or consumed with barbecued meats [2]. Due to its high edible value, it was reported to be grown widely in South Korea, including Kangwan, Jeollabuk, Gyeongsangbuk, Kyonggi, Jeollanam, Chungchongbuk, Chungcheongnam and Gyeongsangnam province [3, 5].

In addition to its edible value, Perilla has been reported to have the potential to be a medical resource [69]. For example, a new substance Pf-gp6 extracted from Perilla was proved to inhibit the replication of HIV-1 [7]. The leaf extract of Perilla was reported to impede SARS-CoV-2 by direct virus inactivation [6]. In brief, Perilla is a very popular plant with high economic value.

In Korea, some pathogens have been found to infect Perilla, resulting in different diseases. Ramularia coleosporii was reported to induce leaf spot on Perilla [10] and Corynespora cassiicola caused stem blight in Perilla [11]. Recently, diseases resulting from plant viruses have been reported in Korea. In Yeongcheon city, Cucumber green mottle mosaic virus (CGMMV) caused mosaic and malformation on Perilla leaves [12]. In addition, turnip mosaic virus (TuMV) was first found to infect Perilla plant in in the Korea in 2020, inducing mild mosaic and yellowing symptoms [13].

TuMV is known as a brassica pathogen due to its significant damage to brassica crops [14]. Recent research has indicated that TuMV spread from the west to the east across Eurasia from approximately the 17th century CE [15]. In recent years, TuMV has been found continuously to cause diseases in Brassicaceae plants in South Korea, such as Chinese cabbage (Brassica rapa var. pekinensis) and radish (Raphanus sativus) [16, 17].

Investigation of the molecular evolutionary history of TuMV is beneficial for us to study the biological properties of the population of TuMV [18]. Variation in the genomes of viruses results from mutation, recombination, adaptation and selection [1821]. Recombination is one of the main forces that accelerate the adaptation and variability, usually resulting in the emergence of mutants and virus isolates able to overcome resistance [21]. As the development of programs that are utilized to detect recombination based on sequence comparison advances, more and more recombination events in plant RNA viruses have been reported [14, 18, 19].

In this research, the complete genomes of two new TuMV isolates were characterized, full-length infectious cDNA clones of these two isolates were constructed and their infectivity was tested by agroinfiltration on Nicotiana benthamiana and sap inoculation on Chinese cabbage cv. CR Victory and radish cv. Iljin. Moreover, we investigated the affiliation of six newly collected isolates from Korea by phylogenetic analysis. We found for the first time that recombination events have occurred in Korean TuMV isolates, which helped us better understand the evolutionary relationship among Korean isolates of TuMV.

Materials And Methods

Sample collection and plant material

A P. frutescens plant sample with typical TuMV-like symptoms including mosaic and chlorosis was observed in Chuncheon city, South Korea (sample collected by Professor Jin-Sung Hong, Kangwon National University). The N. benthamiana, and Chinese cabbage and radish plants used in this study were incubated in 25oC ± 2oC with 16 hours of light and 8 hours of dark. All the soil used was sterilized before use.

RNA extraction, cDNA synthesis and PCR detection 

For virus detection, total RNAs of plant tissues were extracted using TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA), and then the extracted samples were preserved at -70oC. The cDNAs were produced using LeGene Express 1st Strand cDNA Synthesis System with an oligo dT primer. Then detection was conducted by PCR using TuMV-CP-forward primer (5’-TCT CAA TGG TTT AAT GGT CTG G-3’) and reverse primer (5’-AAC CCC TTA ACG CCA AGT AAG-3’) [22].

Construction of full-length clones of TuMV 

To obtain infectious clones, we performed full-length PCR. The PCR mixture of 50 μl was composed of 2 μl of the template cDNA, 25 μl of 2 x PCR buffer for KOD FX Neo, 10 pmol of forward primer containing ApaI site and T7 RNA polymerase promoter sequence (5’-GAG GGG CCC TAA TAC GAC TCA CTA TAG GAA AAA TAT AAA AAC TCA ACA CAA CAT ACA CAA AAC G), 0.4mM dNTPs, 10 pmol of reverse primer containing XmaI site (5’-GAG CCC GGG TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTC CCT TGC ATC CTA TCA AAT G)[17], 1 μl of Taq polymerase (KOD FX Neo, Toyobo, Osaka, Japan). A cDNA template of TuMV that has been successfully amplified before was used as a positive control. Conditions of full-length PCR were 94 °C for 2 min followed by 5 cycles of 10 s at 98 °C, 30 s for annealing at 59 °C, 6 min for extension at 68°C, and then by 30 cycles of 10s at 98 °C, 30s at 65°C, 6 min at 68°C, and finally incubation at 4 °C. Analysis of full-length PCR products was conducted by 0.8% agarose gel electrophoresis with dye incorporated in the gel. The PCR product was digested by ApaI and XmaI and subsequently cloned into the binary vector pJY which was digested by the same enzymes [23, 24]. The recombinants were screened by colony PCR and double enzyme digestion. Then the recombinant plasmids were transformed into E.coli competent cell (DH5a).

Agrobacterium infiltration and sap inoculation

The recombinant plasmids were transformed into Agrobacterium tumefaciens GV2260. The colonies of each clone were incubated on LB plates supplemented with Kanamycin and Rifamycin and then the Agrobacterium cells collected from fresh plates were diluted to approximately OD600 = 0.6 in infiltration buffer (10 mM MES, 10 mM MgCl2, and 150 μM acetosyringone). N. benthamiana plants inoculated with constructed clones were incubated in a growth chamber at 24-26oC (16/8h, light/dark cycle) [17]. Leaves of the infiltrated N. benthamiana plants with symptoms were used to inoculate Chinese cabbage cv. CR Victory and radish cv. Iljin as described previously [17]. 

Sequencing of TuMV infectious cDNA clones 

After the infectivity of each full-length cDNA clone was assessed by agroinfiltration, the full-length clones that have infectivity were sequenced by BIONEER CORPORATION (Daejeon, South Korea). Sequencing was initiated from each terminus using vector specific primers, and continued using primers designed from the obtained sequences (Table 1). The complete genome sequences were compared and assembled in DNAMAN software (Version 5.2, Lynnon BioSoft).

Construction of phylogenetic tree.

The Maximum-likelihood method was utilized to construct phylogenetic tree with 1000 bootstrap replicates in MEGA software (version 7.0). The complete genome sequences of TuMV strains utilized in this study were collected from NCBI (Table 2), including isolates previously reported in South Korea [16, 17, 25] and 6 newly collected isolates (two each from Perilla, radish, and Chinese cabbage)

Recombination analysis

Firstly, complete genome sequences of all Korean TuMV isolates were aligned by clustal X in MEGA 7.0. Recombination analysis was performed using RDP4 software package and employed seven detection methods: RDP [26], GENECONV [27], Bootscan [28], Maxchi [29], Chimaera [30], SiScan [31], and 3SEQ [32]. Recombination events were noted if supported by at least four different methods (p-values < 1.0x10 -6) [18].

Results

Construction of full-length cDNA clones 

The full-length PCR products amplified from Perilla were evaluated by gel electrophoresis (Fig 1A), and then the PCR products were digested using ApaI and XmaI. Subsequently, the digested product was ligated with the pJY vector which was treated with the same enzymes. Colony PCR was performed to screen the colonies harboring recombinants (Fig 1B), and finally we obtained 5 positive colonies which were also confirmed by double enzyme digestion, namely KPF-1, KPF-2, KPF-3, KPF-4 and KPF-5 (Fig 1C).

Results of agroinfiltration test and sap inoculation

At 4 days post inoculation (dpi), we observed weak leaf curling symptoms on the top leaves of N. benthamiana plants inoculated by KPF-2 while no symptoms were observed in plants infiltrated with other isolates. At 6 dpi, plants inoculated by construct KPF-1 also showed symptoms. Symptoms were further recorded at 7, 10 and 14 dpi (Fig 2A). Finally, infectivity was only confirmed for full-length cDNA clones KPF-1 and KFC-2. KPF-2 was shown to cause symptoms more quickly and induced obvious chlorosis which was not induced by KPC-1. The infection was confirmed by RT-PCR as described previously, and the RT-PCR result was consistent with their symptom development (Table 3).

For sap inoculation, both isolates infected radish cv. Iljin systemically, causing mild mosaic symptoms, while neither isolate could infect Chinese cabbage cv. CR Victory systemically (Fig 2B).

Nucleotide and amino acid sequence analysis

The genomes of isolate KPF-1 and KPF-2 are both composed of 9832 nucleotides excluding the poly (A) tail, and the genome is predicted to encode a polyprotein of 3164 amino acids. However, the complete genome size of TuMV isolates we obtained previously was 9833 bp. By contrast, the complete genome sequences of the new isolates reported here lack a base in the 3’ untranslated region (at 9753 nt). The two isolates shared 99.92% identity in nucleotide sequence and 99.84% in amino acid sequence. Alignment analysis revealed that there are 8 nucleotide differences between their genomes which are located in P1 (nt 579), HC-Pro (nt 1279 and 2201), P3 (nt 3528 and 3653), CI (nt 4419), VPg (nt 6233) and NIa-Pro (nt 6597) respectively. Amino acid alignment showed 5 differences, in P1 (R150S); HC-Pro (D384N and P691H); P3 (R1175Q); and VPg (E2035G) (Fig 3).

Phylogenetic analysis

Two phylogenetic trees were constructed by using Maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0. The first phylogenetic tree containing isolates collected from a variety of hosts in multiple countries was divided into four branches, which is consistent with previous studies [18, 19] (Fig 4). In addition, the newly collected Perilla isolates (KPF-1 and KPF-2), and radish isolates (KRS-3 and KRS-8) were grouped together and fell in the Basal-BR group. The Chinese cabbage isolates (KBC-1 and KBC-8) were predicted to belong to the World-B group. However, most of the isolates collected from Korea belong to the Basal-BR group. Subsequently, we constructed a second phylogenetic tree which only include Korean TuMV isolates with PVY as the outgroup (Fig 5). Interestingly, four strains previously identified as Basal-BR isolates were clustered with KBC-1and KBC-8 which belonged to the World-B group in the first tree, forming a special branch separated with the other branch composed of only Basal-BR isolates

Recombination analysis

Due to the close correlation between four Basal-BR isolates and two World-B isolates shown by the second phylogenetic tree, we conducted recombination analysis by RDP 4 software, which is a mature and common tool to study the recombination events which have occurred in virus populations. 

As we expected, isolates BE, and three highly similar isolates (HJY1, HJY2, and R007, sharing 99.93% identity in nt) were detected as recombinants supported by all seven detection methods (Fig 6). Three recombination events were indicated. The information for each recombination event is shown in Table 4, with HJY-1 representing the three closely-related isolates HJY1, HJY2, and R007.

Discussion

P. frutescens, a crop with high economic value, has been studied in recent years. With climate change resulting in rising temperatures, diseases caused by viruses have been reported constantly. More and more attention has been paid to the survey and study on the diseases caused by viruses in Perilla plants. In this research, we collected two TuMV isolates from a Perilla sample collected from Kangwon, South Korea. In addition, the complete genome of each isolate was determined, the genomes of both isolates are comprised of 9832 bp. Interestingly, they lack a nucleotide in the 3’ UTR region compared with all TuMV strains we have previously collected in South Korea [16, 17, 23].

In addition, we successfully constructed full-length infectious clones of these two isolates, namely KPF-1 and KPF-2. Their infectivity was evaluated by agroinfiltration on N. benthamiana. Both isolates are able to infect N. benthamiana systemically, but isolate KPF-2 induced symptoms more quickly, usually two days earlier than isolate KPF-1. Amino acid sequence alignment suggested that there are 5 amino acid differences located in P1, HC-Pro (2), P3 and VPg regions. One conserved motif in HC-Pro region has been studied and proved to recruit and employ host ARGONAUTE1 (AGO 1) in the formation of stable virions, which may be involved the efficient systemic infection [33]. The P1 region is the most variable part of the genome [34], it also has been shown to indirectly adjust the RNA silencing suppression activity through reduction of its proteolytic activity which leads to the accumulation of intermediate P1-HCPro [35]. The P3 region also was reported to be an important symptom determinant affecting the host range and cell to cell movement [36, 37]. Our study provided the complete genome sequences of two new TuMV isolates, and we constructed full-length infectious clones to investigate their corresponding symptom phenotypes on N. benthamiana, which is a useful tool in future studies.

Recently, many studies about TuMV have focused on the common recombinants of TuMV and the evolutionary history of the virus [18]. Besides, some phylogenetic studies have emphasized the utilization of non-recombinant sequences to reduce the ‘noise’ caused by recombinants during the evolutionary analysis [14].

In this study, we analyzed the phylogenetic relationship of six new Korean TuMV isolates and determined their respective affiliation with the four major groups. In addition, owing to the close relationship among several Basal-BR isolates and two World-B isolates, we conducted the recombination analysis for Korean TuMV isolates by RDP4. And the recombinant analysis results suggested that recombination events were detected on isolates BE, HJY1 (and also HJY2 and R007), which is supported by seven detection methods (Fig. 6). The recombinant events 2 and 3 could account for the close relationship among Basal-BR isolates BE, HJY1 (plus HJY2 and R007) and World-B isolate KBC-8 (KBC-1). Similar recombinant cases of TuMV have been reported previously, such as the recombination between the Basal-B group and World-B group identified from Australian TuMV isolates [14]. To our knowledge, there are few studies on recombination analysis of Korean TuMV isolates [15], and our analysis provides new insights into the evolutionary relationships among Korean isolates of TuMV.

Declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors

Acknowledgement 

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Golden Seed Project (GSP), Ministry of Agriculture, Food and Rural Affairs (MAFRA)(213006-05-5-SBL20).

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Tables

Table 1. Sequences of the sequencing primers designed and used in this study

No.

Name

Sequence

1

KPF_F1

AGTGCCATTGCGAACCAC

2

KPF_F2

CAAGATCTTCAAGGCGAAG

3

KPF_F3

TGACGGATAGTGAGTTGTCT

4

KPF_F4

AACGGATCACGAAGGCT

5

KPF_F5

ATATCCTCAAGACAAACACC

6

KFP_F6

ACCGAATAATGAGCTGC

7

KPF_F7

ATTCACAGCGTATATTGCT

8

KPF_F8

TCTTGAACCAACTCGACC

9

R1

AATCTCACCACATGCGCTAA

10

R2

TTGGGGAGGTTCCATTCT

11

R-cover

ACATCCAGATGAAAGCAG

12

R3

GTTCGATCATCCATGCGT

13

R4

TTCCATAAATTCCAAGCGGAT

14

R5

AGTTCGCAGTCGATATTTC

15

R6

TTGGTGCTAATCCAGTGTT

16

R7

CATGGAGGTCACACACCT

17

KPF_R8

TAACGTTTTATTCCATTTGCC

18

KPF_R9

ATACTCGCTTGCTGTGAG

 

Table 2. Details of the TuMV isolates analyzed in this study

No.

Accession Number

Isolates

Original Host

Location

1

AB701698

BEL1

Rorippa nasturtium-aquaticum

Belgium

2

AB093611

BZ1

Brassica oleracea

Brazil

3

D10927

Q-Ca

Brassica napus

Canada

4

KF246570

ZH1

Phalaenopsis sp

China

5

AB252106

CHZJ26A

Brassica campestris

China

6

AB093627

HRD

Raphanus sativus

China

7

AB252103

CH6

Raphanus sativus

China

8

AB252107

CZE5

Brassica oleracea

Czech Republic

9

AB701703

DNK3

Brassica rapa

Denmark

10

AB701708

FRA2

Brassica napus

France

11

NC 001616

Potato virus Y

Solanum spp.

France

12

AB701697

ASP

Allium sp.

Germany

13

AB701734

TIGA

Tigridia sp.

Germany

14

AB701735

TIGD

Tigridia sp.

Germany

15

AB701700

DEU2

Raphanus sativus

Germany

16

AB701699

DEU1

Unknown

Germany

17

AB252117

GRC42

Wild Allium sp.

Greece

18

AB701696

GK1

Matthiola incana

Greece

19

AB701719

HUN1

Alliaria petiolata

Hungary

20

AB440238

IRNTra6

Rapistrum rugosum

Iran

21

AB440239

IRNSS5

Sisymbrium loeselii

Iran

22

AB093602

IS1

Allium ampeloprasum

Israel

23

AB701720

ITA1A

Brassica ruvo

Italy

24

AB093598

AI

Alliaria officinalis

Italy

25

AB701721

ITA2

Cheiranthus cheiri

Italy

26

AB701725

ITA8

Abutilon sp.

Italy

27

AB093600

ITA7

Raphanus sativus

Italy

28

AB093601

Cal1

Calendula officinalis

Italy

29

AB252125

KWB779J

Brassica rapa

Japan

30

MG200170

KBJ5

Raphanus sativus

Korea

31

MG200169

KBJ4

Raphanus sativus

Korea

32

MG200168

KBJ3

Raphanus sativus

Korea

33

MG200167

KBJ2

Raphanus sativus

Korea

34

MG200166

KBJ1

Raphanus sativus

Korea

35

KX674727

HJY1

Raphanus sativus

Korea

36

KX674728

HJY2

Raphanus sativus

Korea

37

KX674729

KIH1

Raphanus sativus

Korea

38

KX674730

KIH2

Raphanus sativus

Korea

39

KX674731

GJS1

Raphanus sativus

Korea

40

KX674732

GJS2

Raphanus sativus

Korea

41

KX674733

GJS3

Raphanus sativus

Korea

42

KX674734

GJS4

Raphanus sativus

Korea

43

KY111268

SW1

Raphanus sativus

Korea

44

KY111267

SW2

Raphanus sativus

Korea

45

KX674726

BE

Raphanus sativus

Korea

46

KY111274

DJ1

Raphanus sativus

Korea

47

KY111273

DJ2

Raphanus sativus

Korea

48

KY111272

DJ3

Raphanus sativus

Korea

49

KY111271

DJ4

Raphanus sativus

Korea

50

KY111270

DJ5

Raphanus sativus

Korea

51

KY111269

DJ6

Raphanus sativus

Korea

52

KU140420

R007

Raphanus sativus

Korea

53

KU140421

R041

Raphanus sativus

Korea

54

KU140422

R65

Raphanus sativus

Korea

55

MZ570590

KPF-1

Perilla frutescens

Korea

56

MZ570591

KPF-2

Perilla frutescens

Korea

57

MW556024

KBC-1

Brassica rapa

Korea

58

MW556025

KBC-8

Brassica rapa

Korea

59

MW556026

KRS-3

Raphanus sativus

Korea

60

MW556027

KRS-8

Raphanus sativus

Korea

61

DQ648591

CAR37A

Cochlearia armoracia

Poland

62

AB701731

POL2

Papaver somniferum

Poland

63

AB701728

POL1

Brassica napus oleifera

Poland

64

AB701729

PRT1

Brassica oleracea acephala

Portugal

65

AB093606

RUS1

Armoracia rusticana

Russia

66

AB362513

TUR9

Raphanus sativus

Turkey

67

AB701717

GBR83

Brassica oleracea

UK

68

AF169561

UK1

Brassica napus

UK

 

Table 3. Results of RT-PCR detection assays of agro-infiltrated Nicotiana benthamiana plants

Clone

N. benthamiana

Number tested

KPF-1

+

3

KPF-2

+

3

KPF-3

-

2

KPF-4

-

2

KPF-5

-

2

 

Table 4.     Recombination events detected among Korea TuMV isolates

Event number

Recombinant

Major parent

Minor parent

Type of     'recombinant'

Detection methods *

 

R

G

B

M

C

S

T

p-value

1

BE

KBC-8

R65

World-B x Basal-BR

+

+

+

+

+

+

+

2.414x10-130

2

HJY1

GJS

KBC-8

Basal-BR x World-B

+

+

+

+

+

+

+

6.946x10-103

3

BE

R65

KBC-8

Basal-BR x World-B

+

+

+

+

+

+

+

7.22x10-64

* R = RDP; G = GENECONV; B = Bootscan; M = Maxchi; C = Chimaera; S = Siscan; T = 3Seq.