The autophagy pathway participates in resistance to tomato yellow leaf curl virus infection in whiteflies

Abstract

Macroautophagy/autophagy plays an important role against pathogen infection in mammals and plants. However, little has been known about the role of autophagy in the interactions of insect vectors with the plant viruses, which they transmit. Begomoviruses are a group of single-stranded DNA viruses and are exclusively transmitted by the whitefly Bemisia tabaci in a circulative manner. In this study, we found that the infection of a begomovirus, tomato yellow leaf curl virus (TYLCV) could activate the autophagy pathway in the Middle East Asia Minor 1 (MEAM1) species of the B. tabaci complex as evidenced by the formation of autophagosomes and ATG8-II. Interestingly, the activation of autophagy led to the subsequent degradation of TYLCV coat protein (CP) and genomic DNA. While feeding the whitefly with 2 autophagy inhibitors (3-methyladenine and bafilomycin A1) and silencing the expression of Atg3 and Atg9 increased the viral load; autophagy activation via feeding of rapamycin notably decreased the amount of viral CP and DNA in the whitefly. Furthermore, we found that activation of whitefly autophagy could inhibit the efficiency of virus transmission; whereas inhibiting autophagy facilitated virus transmission. Taken together, these results indicate that TYLCV infection can activate the whitefly autophagy pathway, which leads to the subsequent degradation of virus. Furthermore, our report proves that an insect vector uses autophagy as an intrinsic antiviral program to repress the infection of a circulative-transmitted plant virus. Our data also demonstrate that TYLCV may replicate and trigger complex interactions with the insect vector.

Keywords: Bemisia tabaci; Tomato yellow leaf curl virus; autophagy; begomovirus; interaction; virus transmission; whitefly.

Figure 1.

TYLCV infection induces autophagy in whiteflies. (A) Immunoblot analysis of whiteflies infected with TYLCV for 24 h and transferred to cotton for 120 h. ATG8-I (16 kDa) is observed in both viruliferous and nonviruliferous whiteflies, and ATG8-II (14 kDa) is induced only in the viruliferous whiteflies. Midguts of nonviruliferous (B) and viruliferous (C) whiteflies were fixed and immunofluorescence labeled with anti-ATG8 antibody and secondary antibody conjugated to Dylight 549 (red). Blue indicates DAPI staining of the nuclei. Twenty midguts of viruliferous whiteflies were measured and 95% of them were positive. A representative image is shown, Bar: 50 μm. TYLCV infection induces autophagosome formation as measured by electron microscopy (D to F). Representative images are shown for nonviruliferous (D, Bar: 1 μm) and viruliferous whiteflies (E and F, Bar: 0.5 μm). The initial autophagic vacuole (AVi)/autophagosome can be identified by its rough endoplasmic reticulum, and a double membrane (E). The multimembrane structure in the degradative autophagic vacuole (AVd)/autolysosome can be observed as well (F). Relative expression level of Atg3 (G), Atg9 (H) and Atg12 (I) were tested by qRT-PCR and ACTB was used as the internal control (*, P < 0.05; **, P < 0.01).

Figure 2.

Time course of autophagy activation in response to TYLCV infection. (A) Accumulation of TYLCV CP and autophagy activation in whiteflies that were infected for 6 h and transferred to cotton for 0 to 288 h. The autophagic flux were also monitored by detecting the turnover of the autophagic receptor and substrate SQSTM1/p62. (B) TYLCV DNA was detected by qPCR (Significant differences are indicated with different letters, P < 0.05). (C to E) TYLCV genes (V1, V2 and C3) were detected by qRT-PCR (Significant differences are indicated with different letters, P < 0.05). (Fto H) Relative expression levels of 3 autophagy genes (Atg3, Atg9 and Atg12) were detected by qRT-PCR (*, P < 0.05; **, P < 0.01).

Figure 3.

Dynamics of TYLCV and autophagy in the gut of the whitefly. Whiteflies were infected with TYLCV for 6 h on tomato and then transferred onto cotton for 0 to 288 h. Guts of viruliferous whiteflies were dissected, fixed and labeled with anti-CP (A) and anti-ATG8 (B) antibodies. Bar: 20 μm. Blue indicates DAPI staining of the nuclei. For each time point, 20 midguts were dissected and similar trend was observed. (C) Localization of CP and ATG8 on the same sample.

Figure 4.

Effects of inhibiting autophagy on TYLCV. (A) TYLCV CP and ATG8 of whiteflies treated with 3-MA (+) or without 3-MA (−) were detected by western blot. (B) TYLCV DNA was detected by qPCR (Significant differences are indicated with different letters, P < 0.05). (Cto E) TYLCV gene expression (V1, V2 and C3) were also detected by qRT-PCR (Significant differences are indicated with different letters, P < 0.05). (F) TYLCV CP, ATG8 and SQSTM1/p62 in whiteflies treated with (+) and without BAF (−) were detected by western blot. (G) The effect of BAF treatment on the accumulation of TYLCV CP at 48 h was detected by immunofluorescence. (H) The effect of BAF treatment on the accumulation of the TYLCV genome. TYLCV DNA was detected by qPCR at different time points (significant differences are indicated with different letters, P < 0.05). (I) Silencing of whitefly Atg3 and Atg9 by feeding double-strand RNA (dsRNA). The expression of Atg3 and Atg9 genes was monitored by qRT-PCR (significant differences are indicated with different letters, P < 0.05). (J) The level of ATG8, SQSTM1/p62 and TYLCV CP was then analyzed by immunoblot. (K) The effect of silencing Atg3 and Atg9 on the accumulation of the TYLCV genome. TYLCV DNA was detected by qPCR (Significant differences are indicated with different letters, P < 0.05).

Figure 5.

Effect of rapamycin on TYLCV DNA, CP and gene expression. (A) TYLCV CP and ATG8 of whiteflies induced with rapamycin (+) or without rapamycin (−) were detected by western blot. (B) TYLCV DNA was detected by qPCR (Significant differences are indicated with different letters, P<0.05). (C to E) TYLCV gene expression (V1, V2 and C3) were also detected by qRT-PCR (significant differences are indicated with different letters, P < 0.05).

Figure 6.

TYLCV retention in whiteflies treated with 3-MA or rapamycin. (A) The whiteflies treated with 3-MA or rapamycin feeding on TYLCV-infected tomato plants for 48 h and then transferred to cotton plant. Ten whiteflies were collected at every time point as shown in the figure after transferring to cotton plants. PCR analyses showed that percentage of samples with viral DNA (significant differences are indicated with different letters, P < 0.05). (B) The amount of virus in each tomato plant. The level of TYLCV is each tomato plant was measured by qPCR and normalized with tomato actin. Statistical analysis was done with the Mann-Whitney test. The horizontal line depicts the medians. Significant differences are indicated with different letters, P < 0.05.

Figure 7.

Autophagy activation in response to TYLCCNV infection. (A) Accumulation of TYLCCNV CP and autophagy activation in whiteflies which were infected for 6 h and transferred to cotton for 0 to 120 h. (B) TYLCCNV DNA was detected by qPCR (significant differences are indicated with different letters, P < 0.05). (C) Midguts of viruliferous whiteflies were dissected, fixed and labeled with anti-ATG8 antibodies. The formation of autophagosomes (red) was monitored in TYLCCNV-infected guts from 0 to 120 h.

Figure 8.

Viral replication and activation of the autophagy pathway. (A) Autophagy activation in whiteflies which were infected for 6 h with PalCuCNV and transferred onto cotton for 0 to 120 h. (B) Formation of autophagosomes in guts of PalCuCNV-infected whiteflies was monitored from 0 to 120 h. (C) PalCuCNV DNA was detected by qPCR (significant differences are indicated with different letters, P < 0.05). (D) Autophagy activation in greenhouse whiteflies which were infected with TYLCV for 6 h and transferred onto cotton for 0 to 120 h. (E) The formation of autophagosomes in guts of TYLCV-infected greenhouse whiteflies from 0 to 120 h. (F) The amount of TYLCV DNA in greenhouse whiteflies was detected by qPCR (significant differences are indicated with different letters, P < 0.05).