The bunyavirus nonstructural protein NSs suppresses plant immunity to facilitate its own transmission by improving vector insect performance

Pandemics of vector-borne human and plant pathogens often rely on the behaviors of their arthropod vectors. Arboviruses, including many bunyaviruses, manipulate vector behavior to accelerate their own transmission to vertebrates, birds, insects, and plants. However, the molecular mechanism underlying this manipulation remains elusive. Here, we report that the non-structural protein NSs of orthotospovirus (order Bunyavirales, family Tospoviridae), is a key viral factor that indirectly modifies vector preference and increases vector performance. NSs suppresses the biosynthesis of volatile monoterpenes, which serve as repellents of the vector Western flower thrips (WFT, Frankliniella occidentalis) instead of using its known silencing suppressor activity. NSs directly interacts with and relocalizes the jasmonate (JA) signaling master regulator MYC2 and its two close homologs, MYC3 and MYC4, to disable JA-mediated activation of terpene synthase genes. The dysfunction of the MYCs subsequently attenuates host defenses, increases the attraction of thrips, and improves thrips fitness. These findings elucidate the molecular mechanism through which a bunyavirus manipulates vector behaviors and therefore facilitate disease transmission. Our results provide important insights into the molecular mechanisms by which tospoviruses NSs counteracts host immunity for pathogen transmission. Author summary Most bunyaviruses are transmitted by insect vectors, and some of them can modify the behaviors of their arthropod vectors to increase transmission to mammals, birds, and plants. NSs is a non-structural bunyavirus protein with multiple functions that acts as an avirulence determinant and silencing suppressor. In this study, we identified a new function of NSs as a manipulator of vector behavior, independent of its silencing suppressor activity. NSs manipulates jasmonate-mediated immunity against thrips by directly interacting with several homologs of MYC transcription factors, the core regulators of the jasmonate-signaling pathway. This hijacking by NSs enhances thrips preference and performance. Many human- and animal-infecting members of the Bunyaviridales also encode NSs and could manipulate vector behavior to accelerate their own transmission. Therefore, our data support the hypothesis that the NSs protein may play conserved roles among various members of the Bunyaviridales in the modification of vector feeding behavior that evolved as a mechanism to enhance virus transmission.

In this study, we identified the NSs protein from thrips-borne TSWV as a viral factor 98 that attracts its insect vector. NSs suppresses the JA signaling pathway in the host 99 plant by directly interacting with MYCs, key regulators of the JA signaling pathway, 100 to reduce host defense responses against thrips; this process functions independently 101 of its virus-silencing suppressor activity. Our results establish a molecular mechanism 5 102 underlying how TSWV establishes a mutualistic relationship with its thrips vector by 103 targeting the activities of plant MYC proteins.

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TSWV infection induces a terpene-dependent preference in thrips vector 106 We first investigated the indirect effect of TSWV infection on the behavioral  genes. However, there was no significant difference in the emissions of the 132 monoterpene D-limonene or the sesquiterpene α-bergamotene (Fig 1C and S1A Fig.).

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To examine whether plant monoterpenes whose levels are reduced in response to 134 TSWV infection play a role in plant-WFT interactions, we performed two-choice 135 assays to compare the choice of non-viruliferous WFT for these monoterpenes vs. the 136 solvent control hexane. As expected, α-pinene and β-pinene has a repellent effect on 137 WFT ( Fig 1D).

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NbTPS5 encodes a monoterpene synthase that produces terpenoids that are 139 repellent to WFT 140 Because several TPS genes in N. benthamiana were severely repressed by TSWV 141 infection, we investigated their roles in plant resistance to WFT. RT-qPCR analysis 142 revealed that the expression levels of TPS genes were very low and did not notably 143 change during the first 24 h of WFT infestation. However, the expression levels of 144 NbTPS5 and NbTPS38 sharply increased at 48 h of infestation (Fig 2A). These results 145 indicate that NbTPS5 and NbTPS38 function in defense responses against WFT 146 infestation in N. benthamiana. We then asked whether NbTPS5 and NbTPS38 147 proteins are responsible for the biosynthesis of WFT repellents α-pinene and β-pinene.

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NbTPS38 is likely a sesquiterpene synthase, like NaTPS38 [23], as these proteins 149 share 91% identity (S1B Fig.). Therefore, we focused on the enzyme activity of 7 150 NbTPS5. We produced recombinant NbTPS5 and incubated the purified protein with 151 its possible substrate, geranyl diphosphate (GPP), for 30 min. Gas 152 chromatography-mass spectrometry (GC-MS) of the enzyme products showed that 153 β-pinene and D-limonene are two monoterpene products of NbTPS5 in vitro (Fig 2B). 154 These results indicate that NbTPS5 is a key virus target gene in the host terpene 155 biosynthesis pathway.

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Our data demonstrated that the tospovirus TSWV increases the attraction of insect 158 vector WFT to its host plant by inhibiting terpene synthase in the host. Next, to 159 explore the protein(s) in TSWV that manipulate viral vector host choice, we selected  RT-qPCR analysis indicated that NbTPS5 was significantly induced by NSm and Ncp 166 but weakly repressed by NSs compared to the YFP control, suggesting that NSs 167 mediates the repressive effect of TSWV on NbTPS5 expression ( Fig 3A). We    Fig.). TSWV represents the American-and Tomato zonate spot virus 227 (TZSV) represents the Euro/Asian-type tospoviruses. We further confirmed this 228 conserved interaction between TZSV NSs and AtMYC2 (S4 Fig.) [29].

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The similarity of these protein interactions prompted us to identify the NSs domain  Thus, we hypothesized that AtMYC2, which interacts with virulence factor NSs, is 240 involved in the viral-induced, volatile-dependent attraction of WFT to the host plant.

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To validate this hypothesis, we performed a GUS staining assay using two transgenic

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To analyze the effects of AtMYC2 and AtTPS10 on the feeding preferences of thrips, 247 we performed two-choice assays using myc2-1, tps10-1, and wild-type Col-0 11 248 Arabidopsis. As shown in Fig 5B, the myc2-1 and tps10-1 mutants were more 249 attractive to WFT than wild type. We also tested the effect of myc234 on host 250 preference, finding that WFT preferred to feed on this mutant over wild type (Fig 5B).

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AtTPS10 encodes a monoterpene synthase, which produces β-ocimene and small 252 amounts of cyclic monoterpenes [32]. We therefore carried out a two-choice assay of 253 β-ocimene to examine whether the attraction of tps10 is terpene-dependent. As 254 expected, β-ocimene had a strong repellent effect on WFT (Fig 5C). These results 255 indicate that AtMYC2 is essential for terpene-dependent immunity against the thrips TSWV host (Fig 1A). We demonstrated that TSWV manipulates this process to affect 280 the behavior of the WFT vector. The volatile terpenoids emitted by virus-infected N. 281 benthamiana plants contained fewer repellents to WFT than healthy plants ( Fig 1C).

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Arabidopsis displayed similar levels of terpene-dependent attraction to WFT (Fig 5C). another monoterpene whose production was also repressed by TSWV infection in our 291 study (Fig. 1C), inhibits the growth of WFT [33,34]. We found that the production of 292 the monoterpene synthase, NbTPS5, was greatly suppressed by TSWV infection. This  TSWV-host interaction (Fig 1D and Fig 2B). Moreover, TSWV infection reduced the 298 expression of various TPSs, including NbTPS5. In addition to NbTPS5, NbTPS38 was 299 also downregulated by TSWV infection (Fig 1B). Interestingly, the likely 300 sesquiterpene synthase gene NbTPS38 was downregulated in response to NSm and 301 Ncp, as well as NSs, supporting the notion that multiple proteins contribute to the 302 complex tripartite TSWV-WFT-plant interaction (S2B Fig.). The expression of 303 MYC2-TPS10, which functions in the monoterpene defense pathway in Arabidopsis, 304 was also attenuated in response to TSWV to benefit WFT (Fig 5) [20,32]. The   defense system by suppressing MYC proteins (Fig 4B). The expression of NSs in 347 Arabidopsis is sufficient to promote WFT performance (Fig 5D). Interestingly, the 348 vector manipulation behaviors of both 2b of Cucumber mosaic virus and NSs of 349 TSWV are independent of their silencing suppressor activity (Fig 3B and Fig 4B) [45], 350 these two cellular activities of viral proteins may function in parallel in plant cells.  Terpene synthase enzyme activity 419 The complete open reading frame of NbTPS5 was cloned into the pGST-DC vector. 420 The protein was expressed in E. coli strain BL21, and purified GST protein was used 421 as a negative control. Protein purification was conducted as previously described [23].      Tissue-specific emission of (E)-α-bergamotene helps resolve the dilemma when