Vector Development and Vitellogenin Determine the Transovarial Transmission of Begomoviruses

Vector development and vitellogenin determine the transovarial transmission of begomoviruses

Jing Weia,1, Ya-Zhou Hea,1, Qi Guoa, Tao Guoa, Yin-Quan Liua, Xue-Ping Zhoub, Shu-Sheng Liua, and Xiao-Wei Wanga,2

aMinistry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China; and bInstitute of Biotechnology, Zhejiang University, Hangzhou 310058, China

Edited by Alexander S. Raikhel, University of California, Riverside, CA, and approved May 16, 2017 (received for review January 31, 2017)

The majority of plant viruses are transmitted by insect vectors decades, a number of virus and insect proteins involved in this between hosts, and transovarial transmission of viruses from transmission process have been identified in some virus–vector vector parents to offspring has great significance to their epide- combinations (11–13). However, mechanisms underlying trans- miology. Begomoviruses are transmitted by the whitefly Bemisia ovarial transmission of viruses have rarely been reported, espe- tabaci in a circulative manner and are maintained through a plant– cially for circulative-nonpropagative viruses, because this group of insect–plant cycle. Other routes of begomovirus transmission are viruses is generally believed not able to replicate and transovarially not clearly known. Here, we report that transovarial transmission transmit in their insect vectors. ∼ from female whiteflies to offspring often happens for one bego- Begomoviruses contain the largest known genus of 288 spe- movirus, Tomato yellow leaf curl virus (TYLCV), and may have cies of plant viruses and are generally known to be transmitted contributed significantly to its global spread. We found that TYLCV by the whitefly Bemisia tabaci in a circulative-nonpropagative B. tabaci entry of the reproductive organ of its vector mainly depended on manner (14, 15). The whitefly is now recognized as a complex containing at least 35 cryptic species (16, 17). During the developmental stage of the whitefly ovary, and the transova- the past 30 y, the two most predominant and damaging species of rial transmission of TYLCV to offspring increased with whitefly the B. tabaci complex, Middle East Asia Minor 1 (MEAM1) and adult age. The specific interaction between virus coat protein Mediterranean (MED), which have been commonly referred to (CP) and whitefly vitellogenin (Vg) was vital for virus entry into as biotype B and biotype Q, respectively, have invaded many whitefly ovary. When knocking down the expression of Vg, the countries worldwide and displaced some indigenous whitefly SCIENCES entry of TYLCV into ovary was inhibited and the transovarial species (17). With the global invasion of MEAM1 and MED AGRICULTURAL transmission efficiency decreased. In contrast, another begomovi- whiteflies, many economic crops are at great risk of infection rus, Papaya leaf curl China virus (PaLCuCNV), CP did not interact with begomoviruses (15, 18, 19). Among these viruses, Tomato with whitefly Vg, and PaLCuCNV could not be transovarially trans- yellow leaf curl virus (TYLCV) is one of the most devastating mitted by whiteflies. We further showed that TYLCV could be viral diseases and has spread to more than 50 countries and maintained for at least two generations in the absence of virus- regions (20, 21). Interestingly, although most of the studies infected plants, and the adult progenies were able to infect reported an absence or low frequency of transovarial trans- healthy plants in both the laboratory and field. This study reports mission in begomoviruses, one case reported that TYLCV can be the transovarial transmission mechanism of begomoviruses, and transovarially transmitted at high efficiency (22) (Table S1). it may help to explain the evolution and global spread of some Therefore, much remains to be learned about whether and how begomoviruses. the begomoviruses are vertically transmitted. In this study, we used TYLCV and Papaya leaf curl China begomovirus | transovarial transmission | vector development | virus (PaLCuCNV), a newly isolated begomovirus in China vitellogenin | whitefly Significance aternal transmission of microbes, including viruses, bac- Mteria, microsporidia, and fungi, by arthropods is a universal The majority of plant viruses are transmitted by insect vectors. phenomenon in nature (1–3). Of the ∼700 known plant viruses, Transovarial transmission of virus from female vectors to off- more than 75% are dependent upon arthropod vectors for trans- spring can be very important in maintaining a source of infection mission between hosts, and some of them can be transmitted ver- and therefore has great epidemiological relevance. Identification tically from mother to offspring in a transovarial manner (4, 5). of vector and virus components involved in transovarial trans- Because transovarial transmission controls virus dispersal in space mission can lead to new strategies to combat virus spread. Here, and time and thus has great importance to virus epidemiology, it has we found that the specific interaction between viral coat protein received constant attention from entomologists and virologists (6, and vector vitellogenin determines transovarial transmissibility 7). However, despite its importance, transovarial transmission of of begomoviruses, which have caused great damage to agricul- plant viruses by insects remains uncommon. Depending on the tural production and are generally believed not to be trans- mode of transmission, plant viruses are classified into four categories ovarially transmitted by insect vectors. Our study gives valuable clues for designing strategies to block begomovirus transmission including nonpersistent, semipersistent, circulative-nonpropagative, and provides insights into the evolution and global spread of and circulative-propagative (4). So far, only circulative-propagative begomoviruses. plant viruses, such as reoviruses, rhabdoviruses, and tenuivirus,

have been confirmed to be transovarially transmitted, because Author contributions: J.W., Y.-Z.H., Y.-Q.L., S.-S.L., and X.-W.W. designed research; J.W., transovarial transmission usually requires the replication of viruses Y.-Z.H., Q.G., and T.G. performed research; X.-P.Z. contributed new reagents/analytic in the vector (8). tools; J.W., Y.-Z.H., Y.-Q.L., X.-P.Z., S.-S.L., and X.-W.W. analyzed data; and J.W., Y.-Z.H., There are multiple barriers during the circulative transmission S.-S.L., and X.-W.W. wrote the paper. of plant and animal viruses, including midgut infection barrier, The authors declare no conflict of interest. dissemination barrier, salivary gland escape barrier, and trans- This article is a PNAS Direct Submission. ovarial transmission barrier (9). Passage of viruses through these 1J.W. and Y.-Z.H. contributed equally to this work. barriers requires specific interactions between virus and vector 2To whom correspondence should be addressed. Email: [email protected]. components (10). Identification of putative components could This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lead to new strategies to interdict viral spread. During the past 1073/pnas.1701720114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1701720114 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 (23, 24), to investigate whether begomoviruses can be transovarially observation, the transovarial transmission of TYLCV to vector transmitted by their insect vectors and the virus or vector com- offspring was examined using MEAM1 whiteflies at 1 or 11 DAE, ponents involved in the transovarial transmission process. Our respectively. The adult whiteflies at 1 DAE transmitted TYLCV study demonstrated that TYLCV can be transovarially trans- to the eggs and nymphs of their progenies in a low frequency (8– mitted by the whitefly vector and that whitefly development is a 13%), but no viral DNA was found in the adult progenies (Fig. key factor in determining transmission rate, whereas PaLCuCNV 1B). Notably, the old mother whiteflies at 11 DAE vertically cannot. Furthermore, we found that specific interactions be- transmitted TYLCV to all developmental stages of their progeny tween viral CP and whitefly endogenous vitellogenin (Vg) are efficiently (Fig. 1B). Virus transmission assays showed that none of vital for virus to pass through the transovarial transmission the 24 healthy plants were infected by TYLCV when exposed to barrier. Our study also revealed the importance of transovarial the adult progenies of 1 DAE viruliferous whiteflies, whereas transmission of TYLCV in maintaining a source of virus in the 5 out of 24 plants were infected after inoculation by adult offspring absence of infected plant and its epidemiological relevance in the of 11 DAE viruliferous whiteflies (Fig. 1C). Our data indicate that field, which may help to explain the worldwide spread and out- the age of whitefly is responsible for transovarial transmission break of this virus. efficiency of TYLCV. Results Whitefly Ovary Is a Selective Barrier That Blocks the Transovarial Transmission of PaLCuCNV. Interestingly, PaLCuCNV was never TYLCV Entry in Whitefly Ovary at the Mature Stage of Ovarian detected in the ovary of viruliferous whiteflies (Fig. S1B)orin Development. First, we observed the anatomy of ovary and any developmental stage of the progeny produced by viruliferous ovariole at different developmental phases in female adult MEAM1 whiteflies at 1 or 11 DAE (Table S2). Virus accumu- whiteflies, which has been reported by Guo et al. (25). Briefly, lation experiment revealed that, after a 48-h access acquisition the ovary development could be classified into four stages period (AAP), the abundance of TYLCV in the whole bodies of according to whitefly development: stage I, freshly emerged – – adults was comparable to that of PaLCuCNV, at both 1 and 11 whitefly; stage II, 1 2 d after eclosion (DAE); stage III, 3 10 DAE (Fig. 1D). However, the abundance of TYLCV in ovaries – DAE; and stage IV, 11 14 DAE (Fig. S1A). The ovariole de- of whiteflies at 11 DAE was severalfold that of whiteflies at 1 velopment could be divided into four phases (phases I, II, III, DAE, whereas for PaLCuCNV, the abundance of the virus was and IV) based on oocyte yolk accumulation. The yolk content of low in ovaries of adult whiteflies at both 1 and 11 DAE (Fig. 1E). oocytes gradually increased by phase (Fig. S1A) (25). Then, we We next performed immunofluorescence staining assays to de- investigated the localization of TYLCV and PaLCuCNV in tect the two viruses in midguts (MGs), primary salivary glands ovaries of viruliferous MEAM1 adult whiteflies at different de- (PSGs), and ovaries of whiteflies at 11 DAE, and found that velopmental stages. Interestingly, TYLCV virions were mainly all MGs were TYLCV-positive or PaLCuCNV-positive; and located in ovaries of the more mature stages (stages III and 96% PSGs were TYLCV-positive, and 88% were PaLCuCNV- IV), but not in ovaries of the less mature stages (stages I and positive, indicating that both TYLCV and PaLCuCNV can reach II) (Fig. 1A), indicating that whitefly development may affect MG and PSG effectively. However, although 79% of tested ovaries the transovarial transmission of TYLCV. To substantiate this were TYLCV-positive, none of the ovaries was PaLCuCNV-positive

Fig. 1. Transovarial transmission of two begomoviruses in MEAM1 whitefly. (A) Detection of TYLCV virions in ovaries of viruliferous MEAM1 adult whiteflies at different developmental stages by immunofluorescence. (Upper panels) Immunostaining of TYLCV CP in different stages of ovaries. (Lower panel) Per- centage of ovaries at different stages infected by TYLCV. Stage I, freshly emerged whitefly (n = 30); stage II, 1–2DAE(n = 30); stage III, 3–10 DAE (n = 30); and stage IV, 11–14 DAE (n = 30). (B) Transovarial transmission of TYLCV to offspring via ovum by MEAM1 whitefly. Virus DNA was traced in individual eggs (n = 60), nymphs (n = 60), or adults (n = 60) in the offspring produced by viruliferous adults at 1 or 11 DAE. (C) The infectivity of two groups of adults that developed from eggs produced by viruliferous whiteflies at 1 (n = 24) or 11 (n = 24) DAE. (D and E) Quantitative analysis of TYLCV and PaLCuCNV (PaL) DNA in whole bodies (D) and ovaries (E) of MEAM1 adult whiteflies following a 48-h acquisition access period (AAP) on virus-infected tomato plants starting at 1 and 11 DAE. (F) Localization of TYLCV or PaLCuCNV (PaL) in midguts (MGs), primary salivary glands (PSGs), and ovaries of viruliferous whiteflies at 11 DAE. (A and F) TYLCV and PaLCuCNV virions were detected by use of a mouse anti-CP monoclonal antibody and goat anti-mouse IgG labeled with Dylight 488 (green) secondary antibody. Cell nucleus was stained with DAPI (blue). White arrows indicate mature oocyte. (A–E) Mean ± SEM from three experiments. P < 0.05 [one-way ANOVA, least significant difference (LSD) test].

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1701720114 Wei et al. Downloaded by guest on September 24, 2021 (Fig. 1F and Fig. S1 C–E). These observations clearly imply follicular cells and just started to be absorbed into oocytes (Fig. 2E that the failure of transovarial transmission of PaLCuCNV and Fig. S2B). Then TYLCV virions were detected not only in the is due to nonpenetration of ovary rather than to inefficient space between follicular cells but also in the oocytes of phase III virus intake. ovarioles, when massive Vg accumulated in the space between follicular cells and was rapidly absorbed into the oocytes (Fig. 2E Interaction Between Viral CP and Vector Vg Is Vital for Virus Entry and Fig. S2B). Moreover, TYLCV and Vg colocalized with each into Whitefly Ovary. The above results indicate that virus entry of other in both the space between follicular cells and oocytes (Fig. whitefly ovary mainly relies on viral type and whitefly developmental 2E). These results suggest that the entry of TYLCV virions into stages. As Vg plays a critical role in yolk formation and oogenesis the oocyte shares the same route of Vg transport into the oocyte. (26–29),weassumedthatVgisinvolvedintheentryofTYLCVinto To verify the role of Vg in transovarial transmission of TYLCV, ovary. Interestingly, the expression level of Vg was positively corre- we knocked down the expression of Vg using RNA interference lated with the transovarial transmission efficiency of TYLCV (RNAi). Compared with whiteflies fed with dsGFP (control), Vg (Fig. 2A). We then tested whether TYLCV CP or PaLCuCNV CP mRNA level decreased by 67% in the group fed with dsVg (Fig. interacts with Vg in vivo by coimmunoprecipitation. Both anti- 3A) and Vg protein level also decreased (Fig. S3A). As the ex- TYLCV CP antibody and anti-Vg antibody coimmunoprecipitated pression of Vg was decreased after ingestion of dsVg, the matu- the other interacting proteins, whereas neither anti-PaLCuCNV CP ration of ovaries dissected from the treated whiteflies was delayed antibody nor anti-Vg antibody coimmunoprecipitated the other (Fig. 3B). Meanwhile, the number of eggs laid by female whiteflies proteins (Fig. 2 B and C). In vitro pull-down assay also confirmed after dsVg treatment decreased by 29% compared with the con- that whitefly endogenous Vg was coeluted with GST-fused TYLCV CP, but not with GST-fused PaLCuCNV CP (Fig. 2D). These trol (Fig. 3C). After inoculation on TYLCV-infected plants for findings suggest that the specific interaction between viral CP and 48 h, the whiteflies had similar level of virus in the whole bodies whitefly Vg is vital for virus entry of ovary. when they had been treated with dsVg or dsGFP (Fig. 3D); Next, we traced the entry process of Vg and TYLCV into oo- however, the abundance of TYLCV DNA decreased by 73% in cytes of viruliferous whitefly at 11 DAE. The ovariole of the the ovaries of dsVg-treated whiteflies compared with that of the whitefly is mainly composed of three parts: the tropharium, seg- control (Fig. 3E). Moreover, the overall TYLCV and Vg signals in regating oocyte, and vitellarium (30). The oocyte, located in the dsVg-treated ovaries were lower than those of the control (Fig. S3 center of vitellarium, is surrounded by a single layer of follicular B–I), suggesting that knockdown of Vg expression reduced yolk accumulation into oocytes and impeded the entry of TYLCV into

cells on its surface and has a nutritive cord connected with the SCIENCES

tropharium (30) (Fig. S2A). We did not detect TYLCV virions in the ovary of whitefly. Most importantly, the frequency of TYLCV AGRICULTURAL phase I ovarioles of viruliferous whiteflies when Vg was not de- transmission to eggs was reduced by 90% compared with the tected in the ovarioles (Fig. 2E and Fig. S2B). TYLCV virions control group (Fig. 3F). Furthermore, the transmission rate of were first detected in the space between follicular cells of phase II TYLCV to eggs by the adults decreased by 57% after oral in- ovarioles, when Vg began to accumulate in the space between gestion of anti-Vg antibody, which would bind to Vg and therefore

Fig. 2. Vg is involved in TYLCV entry into whitefly oocyte. (A) The expression patterns of Vg in adult female whiteflies at different developmental stages. Mean ± SEM of three experiments. P < 0.05 (one-way ANOVA, LSD test). (B and C) Coimmunoprecipitation (CO-IP) of Vg with anti-CP monoclonal antibody (B) and vice versa (C) in whitefly crude extracts. (D) B. tabaci endogenous Vg coeluted with GST-fused TYLCV CP but not with GST-fused PaLCuCNV (PaL) CP. (E) Localization of TYLCV and Vg in follicular cells (upper lane) and oocytes (lower lane) of ovarioles at different developmental phases. Cell nucleus was stained with DAPI (blue). For phases II and III, both Vg (red) and CP (green) are shown. Yellow color indicates the overlay of red and green. FC, follicular cells; O, oocyte.

Wei et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 whiteflies at 1 DAE (Fig. 4B). Similarly, PaLCuCNV was never detected in the progeny of viruliferous MED whiteflies at both 1 and 11 DAE (Table S2). Immunostaining showed that both TYLCV and PaLCuCNV can invade the MG and PSG of MED whiteflies. As for the ovaries, nearly 50% of the tested were TYLCV-positive, whereas none was PaLCuCNV-positive (Fig. S6 A–D). Furthermore, Vg and TYLCV CP colocalized in mature ovarioles of viruliferous MED whiteflies (Fig. S6E), suggesting that Vg of MED whitefly is also involved in the entry of ovary by TYLCV.

TYLCV Can Be Maintained for at Least Two Generations by Whitefly Vector. Next, we examined whether TYLCV could be vertically transmitted from the first generation to the following generations. We conducted transovarial transmission assays with the first-generation adults, which developed from eggs deposited on cotton by viruliferous MEAM1 and MED whiteflies at 11 DAE. We found that the first-generation adults could transmit TYLCV to the second-generation progenies through ovum, with 59–65% for MEAM1 whiteflies and 3–35% for MED whiteflies (Fig. 4C). Moreover, the second-generation adults were able to transmit TYLCV to healthy plants, with 4 out of 30 (13%) test plants being TYLC-positive for MEAM1 whiteflies and 1 out of 30 (3%) test plants being TYLCV-positive for MED whiteflies (Fig. Fig. 3. The role of Vg and viral CP in transovarial transmission of virus. 4D). Additionally, field cage trials and field sampling provided (A) Vg mRNA levels after feeding with dsRNAs. (B and C) dsVg treatment evidence of transovarial transmission of TYLCV by MEAM1 and delayed whitefly ovary development (B) and reduced egg production (C)of MED and its relevance to TYLCV epidemiology in the field (Fig. whitefly. For B, sample sizes were 30 females per treatment, and for C, nine S7 and Table S3). replicates per treatment (five females/replicate). (D and E) TYLCV DNA levels in whole bodies (D) and ovaries (E) of whiteflies after feeding with dsRNAs. (F) dsVg treatment reduced transovarial transmission of TYLCV to eggs. (G) Oral ingestion of anti-Vg antibody decreased transmission rate of TYLCV to eggs. (H) The viral DNA level of mutant TYLCV (mTYLCV) and mutant PaLCuCNV (mPaL) in whitefly whole bodies. (I) Transovarial transmission rate of mTYLCV and mPaL via ovum by MEAM1 whitefly. We detected 45– 80 eggs for F, G, and I.(A–I) Mean ± SEM of three independent experiments. P < 0.05 (one-way ANOVA, LSD test).

inhibit CP–Vg interaction, compared with preimmune serum (control)- treated group (Fig. 3G). To examine the role of viral CP in transovarial transmission, we exchanged partial CP sequence of TYLCV with that of PaLCuCNV (Fig. S4A), and observed the transovarial transmission of the mutated TYLCV (mTYLCV) and mutated PaLCuCNV (mPaL) by whiteflies at 11 DAE. Both mTYLCV and mPaL induced typical disease symptoms after agroinoculated into tomato plants (Fig. S4B). After a 48-h AAP, the whiteflies had a similar amount of mTYLCV and mPaL in their whole bodies (Fig. 3H). However, mTYLCV could not be detected in eggs of viruliferous whiteflies, whereas mPaL was transmitted to the eggs in a high frequency (85%) (Fig. 3I). Immunofluorescence analysis of viruliferous whiteflies at 11 DAE showed that nearly 90% of the tested MGs and 80% of the tested PSGs were mTYLCV- and mPaL-positive. For the ovaries, none of the tested samples was mTYLCV-positive, whereas 71% were mPaL-positive (Fig. S5). Overall, these results demonstrate that specific interactions between viral CP and whitefly Vg determine the transovarial transmission of TYLCV. Fig. 4. Transovarial transmission of TYLCV via ovum by MED and Transovarial Transmission of TYLCV and PaLCuCNV by MED Whiteflies. MEAM1 whiteflies. (A) Virus DNA was traced in individual eggs (n = 60), To examine whether this phenomenon also exists in other nymphs (n = 60), and adults (n = 60) in the offspring produced by virulif- whitefly species, we further investigated the transovarial trans- erous MED adults at 1 and 11 DAE. (B) The infectivity of first-generation mission of TYLCV and PaLCuCNV by MED whiteflies, which is adults that developed from eggs deposited by viruliferous MED adult = = another invasive species of the B. tabaci complex (17). MED whiteflies at 1 or 11 DAE; 1 DAE (n 24) and 11 DAE (n 24). (C) Trans- whiteflies of 1 DAE transmitted TYLCV only to the eggs and ovarial transmission of TYLCV to F2 progeny by F1 adults of either MEAM1 or MED at 11 DAE, which developed from eggs deposited on cotton by F0 nymphs of their progeny; however, whiteflies of 11 DAE trans- viruliferous whiteflies at 11 DAE. Eggs (n = 80), nymphs (n = 80), and adults mitted TYLCV to eggs, nymphs, and adults of their progeny = – (n 80). (D) The infectivity of F2 adults that developed from eggs deposited efficiently (67 85%) (Fig. 4A). Whereas 8 out of 24 tested plants on cotton by F1 adults at 11 DAE, which themselves developed from eggs were infected with TYLCV after inoculation by adult progenies deposited on cotton by F0 viruliferous whiteflies at 11 DAE. MEAM1 (n = 30) of 11 DAE viruliferous MED whiteflies, no viral DNA was and MED (n = 30). (A–D) Mean ± SEM of three experiments. P < 0.05 (one- detected after inoculation by progenies of viruliferous MED way ANOVA, LSD test).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1701720114 Wei et al. Downloaded by guest on September 24, 2021 Discussion transovarially transmit TYLCV to their progeny efficiently, and Vertical transmission of viruses from female parent to offspring is the virus can be maintained for at least two generations by an important strategy for viruses to speed up transmission, and is whiteflies in the absence of a host plant (Figs. 1B and 4). More- recognized to play a significant role in viral epidemics (2, 3). over, the adult progenies that developed on virus nonhost plant Transovarial transmission of plant viruses has received constant can infect healthy plants in the field (Fig. S7). With an almost attention since 1933 (5–7). However, most of the reports were invisible size, viruliferous eggs laid by virus-infected whiteflies on focused on circulative-propagative plant viruses, such as reovi- plants can be transported for long distance with plants by human ruses, rhabdoviruses, or tenuiviruses (31–34). Even so, some re- activities. Thus, the transovarial transmission may also contribute search groups have investigated the transovarial transmission of significantly to the global spread of TYLCV as well as the out- circulative-nonpropagative begomoviruses in the past 50 y. How- break of TYLCV disease in the field. ever, whether begomoviruses can be transmitted transovarially is still in great controversy. Although most of the studies reported Methods absence of transovarial transmission (35–39), several studies Transmission of Virus via Ovum by Whitefly. Viruliferous MEAM1 and MED reported the presence of transovarial transmission, but the effi- adult whiteflies at different developmental stages or under different ciencies varied considerably between studies (40–42) (Table S1). treatments were collected in groups of 10 each (female/male = 1:1). For each Our study proved that TYLCV can be effectively transmitted via replicate of a treatment, a group of insects was placed on the lower surface ovary of whitefly adults of elder age, but not by whitefly adults of of a leaf of cotton plant, a nonhost plant of TYLCV and PaLCuCNV, enclosed in a leaf clip cage. The adults were left on the leaf to feed, mate, and ovi- very young age (Fig. 1). Our findings explained why different re- – – sults were obtained in previous experiments on transovarial posit for 72 h at 26 31 °C, 40 60% relative humidity, and a photoperiod of 14:10 h (light/dark). Then, all adults were removed and five eggs were col- transmission of TYLCV in the last half century, as the age of lected from each replicate using disposable sterilized needles (one needle whiteflies used in those experiments was different. The only report for each individual) and stored at –20 °C for detection of virus DNA sub- that showed a very high rate of TYLCV transovarial transmission – sequently. The remaining eggs were left on the leaf to develop. Eighteen actually used 5 8 DAE whiteflies (42), whereas the other two and 25 d later, five nymphs and five adults were collected from each repli- studies with very low frequency used newly emerged whiteflies (40, cate, using disposable sterilized needles (one needle for each individual) and 41). In addition, not all begomoviruses can be transovarially stored at −20 °C for detection of virus DNA subsequently. transmitted (Table S1); therefore, the virus strains used in different experiments may also contribute to the discrepancy. Transmission of TYLCV to Plants by Adult Progenies. For indoor transmission

Vg, the precursor of major yolk protein in most oviparous assays, the adult progenies of viruliferous whiteflies were collected in groups SCIENCES

species and invertebrates, is synthesized mainly by the fat body of 10 (female/male = 1:1) and each group was used to inoculate one un- AGRICULTURAL and secreted into hemolymph, from where it is absorbed into the infected tomato plant. The inoculation was performed on the top second growing oocytes by receptor-mediated endocytosis, and finally leaf of the plant at the 3–4 true-leaf stage (∼3 wk after sowing) for a 48-h serving as nutrition for developing embryos (28, 43, 44). Here, inoculation access period, using a leaf clip cage. Twenty-four uninfected we discovered that the specific interaction between viral CP and tomato plants were used for the first-generation adults and 30 for the whitefly Vg determines the transovarial transmission specificity second-generation adults. The plants were then sprayed with imidacloprid of begomoviruses, and that the binding between TYLCV CP and at a concentration of 20 mg/L to kill all of the whitefly adults and eggs, and Vg is important for TYLCV to overcome the transovarial maintained until symptoms had developed. transmission barriers (Figs. 2 and 3). TYLCV CP and Vg may bind together in the hemolymph and then be transported into the Coimmunoprecipitation Assay. Immunoprecipitation was performed using oocyte via the existing transportation systems for Vg. In contrast, protein G-Sepharose 4 Fast Flow (catalog no. 17-0405-03; GE Healthcare) according to the manufacturer’s instructions. Whitefly protein extracts were another begomovirus PaLCuCNV could not be transovarially · transmitted by whitefly, and PaLCuCNV CP did not interact with prepared in cell lysis buffer (20 mM Tris HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 20 mM β-glycerophosphate, 10 mM NaF, 1 mM PMSF, 1 mM sodium whitefly Vg (Fig. 2 and Table S2). When we replaced partial CP orthovanadate, 10 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mM EDTA). sequence of PaLCuCNV with that of TYLCV, the mutated Monoclonal antibodies anti-CP (51), anti-Vg (52), or anti-Actin (catalog no. PaLCuCNV became transovarially transmissible and mutated E021020-02; EarthOx) and their corresponding preimmune sera (catalog no. TYLCV became nontransmissible (Fig. 3I and Fig. S5). Thus, A7028; Beyotime) as controls were incubated with whitefly protein extracts this part of CP sequence determines transovarial transmissibility. for 10 h at 4 °C, followed by incubating with protein G-Sepharose beads for However, there are 33 different amino acids in the exchanged an additional 2 h at 4 °C. After washing five times with lysis buffer, immu- fragment (Fig. S4A). To identify the essential amino acids that noprecipitated proteins were eluted by boiling in PAGE buffer for 5 min, and are responsible for transovarial transmission, more virus CP then resolved by SDS/PAGE. Each immunodetection was repeated at least mutants with single-amino acid substitution could be generated three times. Incubation with antibodies was followed by ECL Plus Detection to test their transovarial transmission and interactions with Vg. (catalog no. 170-5060; Bio-Rad). Protein G-Sepharose beads bound to anti- Previous study has shown that the transovarial transmission of a Actin antibody or preimmune sera were used as negative controls. propagative RNA virus (Rice stripe virus) is mediated by Vg of its insect vector (31). Moreover, the Babesia parasite in Hae- GST Pull-Down Assay. The fragments of TYLCV and PaLCuCNV CPs were maphysaalis longicornis and the ZAM virus in Drosophila mela- amplified and cloned into pGEX-6p-1 for fusion with GST. Primers are listed in nogaster are dependent on the Vg receptor when transmitted into Table S4. All recombinant proteins were expressed in Escherichia coli strain the oocyte (45, 46). Therefore, the existing transovarial trans- Rosetta and purified. The GST-tag–fused protein, TYLCV CP-GST or PaLCuCNV portation systems for absorbing nutrients in the oviparous spe- CP-GST, was bound to glutathione Sepharose beads (catalog no. 17-5132-01; GE Healthcare) for 3 h at 4 °C, and the mixtures were centrifuged for 5 min cies, including insects, may be a common way used by DNA virus, × RNA virus, and parasites for their maternal transmission. Given at 100 g, and the supernatants were discarded. The nonviruliferous whitefly soluble protein extracts or the His-tag fusion proteins were added the great significance of vertical transmission for microbe ecol- to the beads and incubated for 2 h at 4 °C. After being centrifuged and ogy, evolution, and epidemiology, more investigations on this washed five times with PBS, the beads-bound proteins were eluted by mechanism among other microbe–host associations are needed. ’ boiling in PAGE buffer for 5 min, and then the proteins were separated by TYLCV, one of the world s most devastating pathogens of to- SDS/PAGE gel electrophoresis and detected by anti-Vg antibody. mato, is considered to have spread from the Middle East or the Eastern Mediterranean to the world since the 1980s, and the Gene Silencing by Oral Ingestion of dsRNA. RNA silencing was performed as dissemination process is probably still ongoing (47, 48). The global previously described (53). Briefly, dsRNAs were diluted into 15% (wt/vol) invasion of MEAM1 and MED whitefly together with the move- sucrose solution at the concentration of 300 ng/μL. Approximately 100 adult ment of infected plant material are believed to be the two main whiteflies at 2 DAE were released into each feeding chamber. The tube was factors that are attributed to the rapid spread of TYLCV world- incubated in an insect-rearing room for 48 h. Subsequently, whiteflies were wide (49, 50). In our study, both MEAM1 and MED whitefly can transferred to TYLCV-infected tomato plants and maintained for 48 h, and

Wei et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 then RNA was extracted from 20 female individuals to examine the Vg incubation with anti-Vg monoclonal antibody (1:500) or anti-CP monoclonal mRNA level, and total proteins were extracted from 30 female individuals to (1:500) or polyclonal antibody (1:500) in PBST containing 1% BSA overnight at examine the Vg protein level after dsRNA treatment. The remaining insects 4 °C and then with goat anti-mouse (1:500) or goat anti-rabbit (1:500) sec- were used for quantitative assays and virus transmission tests. Each set of ondary antibody labeled with Dylight 488 (catalog no. LK-GAM4882; Mul- experiment was repeated three times. tiSciences Biotech) or Dylight 549 (catalog no. LK-GAR5492; MultiSciences Biotech) in PBST containing 1% BSA for 1 h at room temperature after ex- Immunofluorescence Assay. For virus localization, ovaries, MGs, and PSGs of tensive washing. The nucleus was stained with 100 nM 4′,6-diamidino-2- female whiteflies at required developmental stages were dissected after a 48-h phenylindole (DAPI) (catalog no. ab104139; Abcam) in PBST for 2 min at AAP on virus-infected plants. For Vg antibody staining, ovaries of whiteflies at room temperature. 11 DAE were dissected and ovarioles were dispersed from ovaries. The specimens were fixed in 4% paraformaldehyde for 1 h at room temperature, ACKNOWLEDGMENTS. We thank Prof. Gong-Ying Ye for providing whitefly and washed in PBST containing 0.1% Triton X-100 three times for 1 h each. Vg antibody. Financial support for this study was provided by the National Then the specimens were blocked in PBST containing 1% BSA (catalog no. Natural Science Foundation of China (31390421) and the National Key A3828; MultiSciences Biotech) for 2 h at room temperature, followed by Research and Development Program (2016YFC1200601).

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