Earth and Life Institute

Applied microbiology
Phytopathology
Our research group focuses on major plant diseases caused by bacteria, fungi or viruses, with the aim to understand the interaction between the pathogen, (the vector) and the plant. Different models involving both temperate and tropical crops, vectors like the protists Polymyxa spp. or aphids, are investigated on crops like banana, cassava, cereals, potato, sugar beet or tomato

Pomoviruses associated with the rhizomania syndrome

Franois Crutzen

CONTEXT
SUGAR BEET CROPPING
Beet necrotic yellow vein virus (BNYVV), a Benyvirus, is responsible for the rhizomania syndrome of sugar beet. BNYVV is transmitted to Chenopodiaceae by an obligatory root endoparasite, the protist Polymyxa betae (Fig. 2). The disease, first described in Italy in 1959, is now reported in most countries growing sugar beet (Fig. 1). In addition, other viruses such as the pomoviruses Beet virus Q (BVQ) and Beet soil-borne virus (BSBV), are frequently found associated with BNYVV. However, unlike the latter, the pathogenicity of BVQ and BSBV has never been clearly demonstrated and their potential role in the rhizomania syndrome remains a matter of debate. With the view of tackling with the specific functioning of BVQ and BSBV, reverse genetic approaches were developed.
Fig.1. World map showing sugar beet growing countries and regions in black and the sugar production in thousands of tons.

RHIZOMANIA PATHOSYSTEM

Electron micrograph of BNYVV particles.

B A

Structures of Polymyxa betae (arrows) in root cells of beet.

Healthy (A) and BNYVV-infected (B) sugar beets.

Fig. 2. The rhizomania pathosystem involves the disease-responsible Beet necrotic yellow vein virus (BNYVV) transmitted to sugar beet by the obligatory root endoparasite Polymyxa betae.

BVQ GENOME
Compared to other closely related viruses, the Beet virus Q coat protein (CP) readthrough (RT) domain is reported to be much shorter, allowing place for two small ORFs coding for proteins of 9 (p9) and 18 kiloDaltons (kDa) (p18) (Fig. 3). As such a genome organization of BVQ has been described for a unique lab strain in 1998, we partially characterized the RNA-2 of ten BVQ strains from Belgium, France, Germany, Hungary, Spain and The Netherlands. The partial sequencing of these distinct BVQ sources from six countries evidenced a longer RT domain for the CP, with three distinct nucleotide additions of 5, 285 and 1 nt being responsible for this rearrangement.
Genome organization of BVQ, as described in 1998
RNA-1
CAP

BSBV FUNCTIONING
For a better understanding of the functionality and pathogenicity of Beet soil-borne virus (BSBV), full-length cDNA clones were constructed for the three genomic RNAs (genome organization identical to BVQ, Fig. 3). A mutant was constructed for the CP RT domain (represented as 2* below). With the aim of assessing their effectiveness and relative contribution to the virus housekeeping functions, transcripts from full-length cDNA clones were inoculated on Chenopodium quinoa and Beta macrocarpa leaves using five genome combinations.

VIRAL INTERACTIONS
Full-length cDNA clones were also constructed for the tripartite genome of BVQ and the infectiousness of transcripts produced in vitro from constructs was tested on C. quinoa leaves. However, BVQ transcripts were not self-infectious... Following the mixed infections reported in sugar beet crops, transcripts of BVQ were then mixed with transcripts from fulllength cDNA clones of BSBV or BNYVV as shown beneath.
mixed with BSBV? BVQ 1+2+3 mixed with BNYVV?

BVQ 1+2+3 & BNYVV 1+2 BVQ 1+2+3 & BSBV 1+2+3 BSBV 1+2+3
C. quinoa

BNYVV 1+2

Symptom expression on the inoculated leaves

C. quinoa B. macrocarpa
no symptoms

BVQ 1+2+3 & BNYVV 1+2+3+4 BNYVV 1+2+3+4

*
MTR
149 kDa

HEL

POL
RT 207 kDa

Common organization of BVQ RNA-2

*
CP
19 kDa RT 95 kDa

Detection of viral RNAs by Northern blot

12*3 123 12 13 23
-C

RNA-2
CAP

CP
19 kDa RT 35kDa

p9

p18

RNA-3
CAP

TGB p53
p13

p20

Characterization of BVQ CP RT domain, for ten viral strains

Longer CP-RT No p9 & p18

Length scale (kb)

Detection of viral RNAs in leaves

C. quinoa
1 2 3 4 5 6
+C

B. macrocarpa
1 RNA-1 2 3 4 5 6
+C

Replication

Structure

Transmission

Movement

Unknown

0.5

1.0

1.5

2.0

RNA-1

Fig. 3. Schematic representation of the BVQ genome, composed of three singlestranded plus-sense RNAs. The structural coat protein (CP) and a readthrough (RT) domain are expressed from RNA-2. The molecular mass is indicated in kiloDaltons (kDa) for each protein under or inside the corresponding ORF. Function(s) attributed or suspected for each protein is/are specified through a color code.

RNA-2 RNA-3
+C

RNA-2 RNA-3

= diluted transcripts
+C

= diluted transcripts

Detection of viral structural proteins in leaves

C. quinoa B. macrocarpa
6 115 kDa CP-RT 19 kDa CP 1 2 3 4 5 6 115 kDa CP-RT 19 kDa CP 1 2 3 4 5

Such special organization of BVQ RNA-2 previously described in 1998 is hypothesized to result from multiple inoculation cycles of this BVQ strain on the lab host Chenopodium quinoa.

Transcripts from full-length cDNA clones of BSBV RNAs-1 and -3 are essential and sufficient for symptom expression on C. quinoa and viral multiplication in planta.

Transcripts from full-length cDNA clones of BVQ were not self-infectious but required the simultaneous presence of BNYVV RNAs-1 and -2 to multiply in planta. Nevertheless, such rescue was not observed when BVQ was co-inoculated with the closely related BSBV.

+C

BS BN BQ RNA-1

BSBV

RNA-2 RNA-3

RNA-1 RNA-2

BNYVV
RNA-3 RNA-4

RNA-1

BVQ

RNA-2 RNA-3

BS BV 123 -B V Q1 BS 23 BV 123 BN YV V12 -B V Q1 BN 23 YV V12 BN YV V12 34-B BN VQ YV 123 V12 34 BV Q1 23 C

CAP