ToBRFV Infects the Reproductive Tissues of Tomato Plants but Is Not Transmitted to the Progenies by Pollination

Abstract

Tomato brown rugose fruit virus (ToBRFV), a newly identified Tobamovirus, has recently emerged as a significant pathogen of tomato plants (Solanum lycopersicum). The virus can evade or overcome the known tobamovirus resistance in tomatoes, i.e., Tm-1, Tm-2, and its allele Tm-2². ToBRFV was identified for the first time only a few years ago, and its interactions with the tomato host are still not clear. We investigated ToBRFV's presence in the reproductive tissues of tomato using fluorescent in situ hybridization (FISH) and RT-PCR. In infected plants, the virus was detected in the leaves, petals, ovary, stamen, style, stigma, and pollen grains but not inside the ovules. Fruits and seeds harvested from infected plants were contaminated with the virus. To test whether the virus is pollen transmitted, clean mother plants were hand pollinated with pollen from ToBRFV-infected plants and grown to fruit. None of the fruits and seeds harvested from the pollinated clean mother plants contained ToBRFV. Pollen germination assays revealed the germination arrest of ToBRFV-infected pollen. We concluded that ToBRFV might infect reproductive organs and pollen grains of tomato but that it is not pollen transmitted.

Keywords: FISH; ToBRFV; Tobamovirus; flower; pollen; seed; tomato.

Figure 1

Structure and organs of a tomato flower.

Figure 2

Representative confocal microphotographs of ToBRFV presence in the vegetative and reproductive organs of tomato. In situ hybridization with a specific ToBRFV DNA probe fluorescently labeled with Cy3. Intact uninfected plants served as control. (A) Leaf surface in control plant, Bar = 50 µm; (B) Leaf surface in infected plant. Note numerous red dots (fluorescent signal) in the epidermal cell and trichomes (t), Bar = 50 µm; (C) Transverse section of the infected leaf. The signal is visible in all leaf tissues and trichomes (t), Bar = 100 µm; (D) Section of the sepals of infected plant; note fluorescent signal (small red dots), Bar = 200 µm; (E) Cross-section of the control uninfected ovary; ovules (arrows) are visible, Bar = 200 µm. (F) Cross-section of the infected ovary; ovules (arrows) are not infected, but ovary walls, pericarp, and placental tissues (p) show fluorescence signal of virus infection. Bar = 200 µm.

Figure 3

Representative confocal microphotographs of ToBRFV presence in the flower organs of tomato. In situ hybridization with a specific ToBRFV DNA probe fluorescently labeled with Cy3. Intact uninfected plants served as control. (A) Transverse section of the anther of control plant. Clean pollen and anther tissues are visible, Bar = 100 µm; (B) Transverse section of the anther of the infected plant. Note fluorescent signal in the anther tissue, pollen is partly infected, Bar = 100 µm; (C) Transverse section and double staining with DAPI and Cy3 of the infected anther. Fluorescence signal (red dots) is visible in several pollen grains, Bar = 200 µm; (D) Transverse section of control style, Bar = 200 µm; (E) Transverse section of infected style. The stigma of the infected flower looks heavily infected. Bar = 200 µm; (F) Pollen grains of control plant, Bar = 200 µm; (G) Close-up of the pollen grain of the control plant, double stained with DAPI and Cy3. Note the absence of red fluorescence signal (viral infection). The pollen nucleus is stained blue (arrow). Bar = 10 µm; (H) Close-up of the pollen grains in the infected plants. Bar = 20 µm; (I) Pollen grains of infected plants, Bar = 50 µm; (J) Close-up of the pollen grain of the infected plant, double staining with DAPI and Cy3. Note red fluorescence signal (viral infection). The pollen nucleus is stained blue (arrow). Bar = 10 µm.

Figure 4

PCR analysis of tomato seeds for ToBRFV presence and infectivity test for seed-associated ToBRFV. (A) RT-PCR detection of ToBRFV in seeds harvested from infected plants. Each lane represents 15 seeds harvested from a different plant. Lanes: (1) Molecular weight markers; (2–11) Seeds harvested from infected plants; (12, 15) empty lanes; (13) seeds harvested from a noninoculated plant; (14) reaction mix; (16) Total RNA from an infected leaf. (B,C) Tobacco leaf (N. tabacum cv. Xanthi–NN) was inoculated with a crude extract from 50 seeds harvested from healthy tomato (B) or from an infected tomato plant (C). Note the presence of local lesions (arrows). ToBRFV’s presence in the developed local lesions was validated by RT-PCR. Pictures were taken 72-hr after inoculation.

Figure 5

Representative confocal microphotographs of ToBRFV presence in germinating pollen grains of tomato. Pollen grains were allowed to germinate for 90 min, followed by DAPI staining and in situ hybridization with a specific ToBRFV DNA probe fluorescently labeled with Cy3. (A) Germination of pollen from control noninfected plant; nuclei stained in blue (DAPI) are visible in the pollen grain and tubes (arrows), Bar = 50 µm; (B) Germination of pollen from infected plant. Note that the infected grains do not germinate (stained in red). In germinating noninfected grains, nuclei are stained in blue (DAPI) and are visible in the grain and pollen tubes (arrows), Bar = 50 µm.