Citrus leaf blotch virus invades meristematic regions in Nicotiana benthamiana and citrus

Abstract

To invade systemically host plants, viruses need to replicate in the infected cells, spread to neighbouring cells through plasmodesmata and move to distal parts of the plant via sieve tubes to start new infection foci. To monitor the infection of Nicotiana benthamiana plants by Citrus leaf blotch virus (CLBV), leaves were agroinoculated with an infectious cDNA clone of the CLBV genomic RNA expressing green fluorescent protein (GFP) under the transcriptional control of a duplicate promoter of the coat protein subgenomic RNA. Fluorescent spots first appeared in agroinfiltrated leaves 11-12 days after infiltration, indicating CLBV replication. Then, after entering the phloem vascular system, CLBV was unloaded in the upper parts of the plant and invaded all tissues, including flower organs and meristems. GFP fluorescence was not visible in citrus plants infected with CLBV-GFP. Therefore, to detect CLBV in meristematic regions, Mexican lime (Citrus aurantifolia) plants were graft inoculated with CLBV, with Citrus tristeza virus (CTV), a virus readily eliminated by shoot-tip grafting in vitro, or with both simultaneously. Although CLBV was detected by hybridization and real-time reverse transcription-polymerase chain reaction (RT-PCR) in 0.2-mm shoot tips in all CLBV-inoculated plants, CTV was not detected. These results explain the difficulty in eliminating CLBV by shoot-tip grafting in vitro.

Figure 1

Outline of the infectious Citrus leaf blotch virus (CLBV) clone clbv3'pr‐GFP expressing the green fluorescent protein (GFP). Grey boxes represent the CLBV open reading frames (ORFs) (227‐kDa polyprotein containing the replicase domains; MP, movement protein; CP, coat protein) and the white box the gfp gene. Arrows indicate transcription of subgenomic RNAs (sg). White triangle represents the promoter of the CP subgenomic RNA, duplicated to express GFP.

Figure 2

Expression of the green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinoculated with the infectious Citrus leaf blotch virus (CLBV) clone clbv3'pr‐GFP. (a, b) Infection foci in agroinoculated leaves at 13 and 16 days post‐inoculation (dpi), respectively. (c) Virus loading into the phloem vessels at different sites of an agroinoculated leaf. (d) Fluorescence focus beside a vein observed in a fluorescence stereomicroscope. Bar, 80 μm (e) Epidermal cells observed by confocal microscopy. Bar, 10 μm. n, nucleus; c, cytoplasm. (f) GFP accumulation in stem, petiole and veins of upper leaves at 18 dpi. (g) Plant systemically infected at 25 dpi. (h, j) Cross‐sections of stem and petiole from an infected plant. (k) Similar petiole cross‐section from an intense fluorescence spot. (i, l) Similar stem and petiole cross‐sections from a healthy plant. Bar, 500 μm. E, external phloem; I, internal phloem; pa, parenchyma; ph, phloem; xy, xylem. (m) Upper leaf of infected plants at 20 dpi. (n) Leaf undergoing the sink–source transition at 20 dpi. (o) Roots of infected (left) and healthy (right) plants. Photographs a–c, f, g and m–o were taken with a digital camera using UV light and a yellow filter, d and h–l with a fluorescence stereomicroscope and e with a confocal laser scanning microscope.

Figure 3

Citrus leaf blotch virus (CLBV) detection by molecular hybridization in infected tissues of Nicotiana benthamiana and citrus plants. (a) Imprints of different tissues from N. benthamiana plants agroinoculated with the CLBV infectious clone clbv3'pr‐GFP showing green (green fluorescent protein, GFP) or red (chlorophyll) fluorescence. (b) Dot‐blot hybridization using total RNA extracted from healthy or CLBV‐infected Citrus clementina (pollen), Mexican lime (shoot tips, ST) or N. benthamiana (ST) plants. (c) Tissue‐print hybridization of petals, ovary or ovules from healthy or CLBV‐infected C. clementina plants. The membranes were hybridized with a digoxigenin (DIG)‐labelled RNA probe specific for the CLBV 3′ untranslated region (UTR). (d) Detection and absolute quantification of genomic RNA copies of CLBV (q‐clbv) or Citrus tristeza virus (CTV) (q‐ctv) in 0.2‐mm shoot tips or bark of Mexican lime plants infected with CLBV, CTV or co‐inoculated with both viruses. –, not detected.

Figure 4

Green fluorescent protein (GFP) detection in meristems and floral organs of Nicotiana benthamiana plants agroinoculated with the infectious Citrus leaf blotch virus (CLBV) clone clbv3'pr‐GFP. (a–c) Shoot apical meristem (SAM). (d–f) Axillary meristem. (g, h) Longitudinal sections of flowers from an infected plant at early developmental stages (g1 and h) and a similar section from a healthy plant (g2). (i–k) Developing ovary, style, stigma and corolla from an infected plant, and similar organs from a healthy plant (p–r). (l, m) Longitudinal section of a mature ovary from an infected plant (l) and a similar ovary after eliminating the carpel (m). (n) Young seeds from infected (left) and healthy (right) plants. (o) Mature seed from a healthy plant. Images (b, c, e, f) were captured using a confocal laser scanning microscope, (a, d) with a light microscope and (g–r) with a fluorescence stereomicroscope. Bars in the latter images, 500 μm.