Callose deposited at soybean sieve element inhibits long-distance transport of Soybean mosaic virus

Abstract

The function of callose and its deposition characteristics at phloem in the resistance to the long-distance transportation of Soybean mosaic virus (SMV) through phloem was studied. Two different methods of SMV inoculation were used in the study, one was direct friction of the virus on seedling leaves and the other was based on grafting scion and rootstock to create different resistance and sensitivity combinations. Veins, petioles of inoculated leaves and rootstock stems were stained with callose specific dye. Results from fluorescence microscope observation, pharmacological test, and PCR detection of SMV coat protein gene (SMV-CP) showed the role of callose in long-distance transportation of SMV through phloem during infection of soybean seedlings. When the inhibitor of callose synthesis 2-deoxy-D-glucose (2-DDG) was used, the accumulation of callose fluorescence could hardly be detected in the resistant rootstocks. These results indicate that callose deposition in phloem restricts the long-distance transport of SMV, and that the accumulation of callose in phloem is a main contributing factor for resistance to this virus in soybean.

Keywords: Callose; Grafting; Long-distance transport; Soybean mosaic virus.

Fig. 1

Callose in different tissues of plants following inoculation with SMV. A Fluorescence from stained callose in leaf vein of Wuxing 2 frictionally inoculated with N3; B micrograph of A under transmission light; C Fluorescence from stained callose in petiole of Wuxing 2 frictionally inoculated with N3; D micrograph of C under transmission light; E callose was not detectable in stem of Wuxing 2 frictionally inoculated with N3; F micrograph of E under transmission light; G callose was not detectable in vein of Wuxing 2 simulating inoculated; H micrograph of (G) under transmission light; I callose was not detectable in petioles of Wuxing 2 simulating inoculated; J micrograph of (I) under transmission light; K: callose was not detectable in stems of the Wuxing 2 inoculated with water; L micrograph of K under transmission light; a, c, e, g, i, k: enlarged pictures of the areas labelled with the arrows in picture (A, C, E, G, I, K)

Fig. 2

Spread of SMV in the incompatible host. A Schematic diagram showing plants inoculated with SMV; B Detection of SMV-CP gene by RT-PCR analysis from different tissues of soybean plants. M: DL2000bp DNA marker; 1: negative control (simulated inoculation with water); 2: positive control (Jihuang 13 leaves inoculated with SC-8); 3: the veins of inoculated leaves; 4: the petiole of inoculated leaves; 5: the stem; 6: non-inoculated upper leaves

Fig. 3

Schematic diagram showing virus inoculation of different resistant genotypes after grafting

Fig. 4

Callose in rootstock stem when scion was inoculated with SMV. A Non-inoculated upper leave of the rootstock; B Scion leaves inoculated with SMV; C Callose fluorescence was detected in stem of rootstock below the grafting site at 3-days after scion nf58 was frictionally inoculated with N3; D micrograph of C under transmission light; E Callose fluorescence was not detected in stem of rootstock above the grafting site at 3 days after scion nf58 was frictionally inoculated with N3; F micrograph of E under transmission light; G Callose fluorescence was not detected in stem base of the rootstock Wuxing 2 at 3-days after scion Jihuang 13 was frictionally inoculated with SC-8; H micrograph of (G) under transmission light; c, e, and g: the enlarged pictures of the areas labeled with arrows in picture C, E, and G

Fig.5

Detection of SMV-CP gene product. A Detection of SMV CP gene on grafted plants; M: DL 2000 bp DNA marker; 1: positive control (Jihuang 13 leaves inoculated with SC-8); 2: negative control (simulating inoculated leaves); 3: scion nf58 leaves after inoculation; 4: Stem of rootstock above the grafting site; 5: Stem of rootstock below the grafting site; 6: non-inoculated upper leaves of rootstock. B SMV-CP gene detection in rootstock when scion nf58 inoculated with N3 was pre-treated with 2-DDG. M:DL2000 bp DNA marker; 1: positive control (Jihuang 13 leaves inoculated with SC-8); 2: negative control (leaves inoculated with water); 3: upper leaves of Wuxing 2; 4: stem of rootstock above the grafting site; 5: stem of rootstock below the grafting site; 6: scion leaves after inoculation. C Western blotting detection of SMV CP. M: Easysee western marker; 1: Leaves of scion after inoculation with SMV; 2: Rootstock stem below the grafting site; 3: Uninoculated rootstock leaves above the grafting site; 4: Stem of rootstock above grafting site