Comparative Plant Transcriptome Profiling of Arabidopsis thaliana Col-0 and Camelina sativa var. Celine Infested with Myzus persicae Aphids Acquiring Circulative and Noncirculative Viruses Reveals Virus- and Plant-Specific Alterations Relevant to Aphid Feeding Behavior and Transmission

ABSTRACT Evidence is accumulating that plant viruses alter host plant traits in ways that modify their insect vectors’ behavior. These alterations often enhance virus transmission, which has led to the hypothesis that these effects are manipulations caused by viral adaptation. However, we lack a mechanistic understanding of the genetic basis of these indirect, plant-mediated effects on vectors, their dependence on the plant host, and their relation to the mode of virus transmission. Transcriptome profiling of Arabidopsis thaliana and Camelina sativa plants infected with turnip yellows virus (TuYV) or cauliflower mosaic virus (CaMV) and infested with the common aphid vector Myzus persicae revealed strong virus- and host-specific differences in gene expression patterns. CaMV infection caused more severe effects on the phenotype of both plant hosts than did TuYV infection, and the severity of symptoms correlated strongly with the proportion of differentially expressed genes, especially photosynthesis genes. Accordingly, CaMV infection modified aphid behavior and fecundity more strongly than did infection with TuYV. Overall, infection with CaMV, relying on the noncirculative transmission mode, tends to have effects on metabolic pathways, with strong potential implications for insect vector-plant host interactions (e.g., photosynthesis, jasmonic acid, ethylene, and glucosinolate biosynthetic processes), while TuYV, using the circulative transmission mode, alters these pathways only weakly. These virus-induced deregulations of genes that are related to plant physiology and defense responses might impact both aphid probing and feeding behavior on infected host plants, with potentially distinct effects on virus transmission. IMPORTANCE Plant viruses change the phenotype of their plant hosts. Some of the changes impact interactions of the plant with insects that feed on the plants and transmit these viruses. These modifications may result in better virus transmission. We examine here the transcriptomes of two plant species infected with two viruses with different transmission modes to work out whether there are plant species-specific and transmission mode-specific transcriptome changes. Our results show that both are the case.


50
Most known plant viruses rely on vectors for transmission to a new host (for example Dietzgen et 51 al., 2016). Insects that feed on phloem sap, such as whiteflies and aphids, are important vectors 52 transmitting at least 500 virus species (Fereres and Raccah, 2015). The high virus transmission 53 capacity is due to their particular non-destructive feeding behavior that allows virus acquisition 54 from and inoculation into the cytoplasm and/or the phloem sap of a new host plant. In fact, aphids 55 alighting on a new plant will first evaluate the potential host for suitability by exploratory 56 intracellular punctures into the epidermis and underlying tissues. If the plant is accepted, aphids 57 plunge their needle-like mouthparts, the so-called stylets, for prolonged feeding phases into the 58 sieve cells whose sap constitutes their principal nutritive source (for review Dáder et al., 2017). 59 Aphids secrete different saliva types during both the probing and the feeding phases that contain 60 effector molecules controlling interactions with the plant and susceptibility (Rodriguez and  Therefore, this transmission mode is also referred to as persistent transmission. 74 The transmission mode of non-circulative viruses such as cucumber mosaic virus (CMV, genus 75 Cucumovirus), that are also transmitted by aphids and other hemipteran vectors, is entirely different 76 (for review Ng and Falk, 2006)). They are mostly acquired and inoculated during early probing While there is overwhelming evidence that some viruses do induce plant phenotype 103 manipulation in ways that are conducive for their own transmission, many significant knowledge 104 gaps remain (discussed by Mauck and Chesnais, 2020). In particular, the mechanisms and pathways 105 by which viruses alter aspects of the host phenotype, and the virus components that are responsible, 106 remain poorly understood (Mauck et al., 2019). 107 In the present study, we addressed these shortcomings and initiated analysis of the effects of two 108 viruses, TuYV and CaMV, belonging to two different transmission categories, on transcriptomic 109 profiles in two susceptible host plant species (Arabidopsis thaliana and Camelina sativa, both 110 family Brassicaceae), and on changes in insect vector feeding behavior and performances. We 111 selected the green peach aphid CaMV isolate Cm1841-Rev (Chesnais et al., 2021), which is a transmissible derivative of isolate 131 Cm1841 (Tsuge et al., 1994), and TuYV isolate TuYV-FL1 (Veidt et al., 1988) were maintained in 132 A. thaliana Col-0 and propagated by aphid inoculation of 2-week-old plants. Growth conditions 133 were as described below. 134  (Veidt et al., 1988)]. Note that the 174

Virus infection and aphid infestation
CaMV reference sequence was extended at the 3'-end by 74 nts from the 5'-terminus to account for 175 its circular genome and allow for mapping reads containing the first and last nucleotides of the 176 linear sequence. In the case of TuYV, some discrepancies with the reference sequence were 177 detected, when the reads were mapped to the viral reference sequence. Therefore, the reads were 178 used to generate a new consensus master genome in the viral quasispecies population. For both 179 viruses, the consensus genome sequences (Supplementary Sequence information S1) were used for 180 (re-)mapping and counting total viral reads as well as viral reads representing forward and reverse 181 strands of the viral genomes (Supplementary Dataset S1). 182

RT-qPCR 183
Expression of Arabidopsis genes was monitored by RT-qPCR analysis. cDNA was synthesized 184 from 10 μg total RNA using AMV Reverse Transcriptase (Promega) and oligo-dT. Real-time qPCR 185 reactions were completed in the LightCycler® 480 instrument (Roche) using the SybrGreen master 186 mix (Roche) and following the recommended protocol. Each reaction (10 µl) included 3 µl of 187 cDNA and 0.5 µl of 10 µM primers (Supplementary Table S1 TuYV) with zero mismatches, and the mapped reads were sorted by polarity (forward, reverse and 213 total) and counted. Viral read counts were then normalized per million of total (viral + non-viral) or 214 plant reads (see Supplementary Dataset S1). 215

Aphid feeding behavior 216
To investigate the effects of TuYV and CaMV plant infections on the feeding behavior of M. 217 persicae, we used the electrical penetration graph technique (EPG) (Tjallingii, 1988

Statistical analyses of aphid behavior and fecundity 242
Data on aphid feeding behavior were analyzed using generalized linear models (GLMs) with a 243 likelihood ratio and the chi-square (χ²) test. Since duration parameters (i.e. probing duration, stylet 244 pathway phase, phloem sap ingestion and salivation) were not normally distributed, we carried out 245 GLMs using a gamma (link = "inverse") distribution. For the "time to first phloem phase", we used 246 the cox proportional hazards model and we treated cases where the given event did not occur as 247 censored.
The assumption of validity of proportional hazards was checked using the functions 248 "coxph" and "cox.zph", respectively (R package "survival"). For aphid fecundity, count data were 249 not normally distributed. Accordingly, we carried out a GLM using a Poisson distribution, a quasi-250 likelihood function was used to correct for overdispersion, and Log was specified as the link 251 function in the model. When a significant effect of one of the main factors was detected or when an 252 interaction between factors was significant, a pairwise comparison using estimated marginal means 253 (R package "emmeans") (p value adjustment with Tukey method) at the 0.05 significance level was 254 used to test for differences between treatments. The fit of all GLMs was controlled by inspecting 255 residuals and QQ plots. All statistical analyses were performed using R software v. 4.0.4 (www.r-256 project.org/).

263
Plant phenotype 264 We used in this study 5-week-old Arabidopsis or Camelina plants that had been inoculated with 265 CaMV or TuYV three weeks before analysis. In both Arabidopsis and Camelina plants, CaMV 266 caused severe leaf curling, mosaic and vein chlorosis as well as dwarfism ( Figure 1). TuYV-267 infected Arabidopsis and Camelina plants were smaller compared to mock-inoculated plants, but 268 showed no leaf deformation or bleaching. Older TuYV-infected Arabidopsis leaves turned purple, 269 probably due to stress-induced anthocyanin accumulation as previously reported for infection of

283
Aphid feeding behavior and fecundity 284 We used EPG to compare aphid probing and feeding behavior on Arabidopsis and Camelina 285 infected or not with CaMV or TuYV (Figure 2a, reported neutral to slightly positive effects of TuYV infection on aphid probing and feeding 300 behavior, and highlighted also host-specific viral effects on plant quality and vector behavior 301 (Chesnais et al., 2019b). 302 Taken together, the significantly reduced time until first phloem ingestion observed on infected 303 Arabidopsis might contribute to a better acquisition of CaMV and TuYV. The other transmission-304 related feeding parameters were only marginally modified on TuYV-infected plants, whereas 305 CaMV infection altered aphid feeding more strongly. The reduced pathway phase and the increased 306 phloem ingestion might also facilitate CaMV acquisition from phloem tissues. These alterations are 307 expected to be detrimental for non-circulative viruses (such as the non-persistent potyviruses) that 308 are acquired during intracellular penetrations occurring in the pathway phase, but lost if the aphid 309 stylets reach the phloem sap (Kloth and Kormelink, 2020). However, this does not apply to CaMV, 310 acquired efficiently from phloem sap as well as mesophyll and epidermis cells (Palacios et al., 311 2002). 312 Infection with CaMV reduced aphid fecundity significantly on both plant host species ( Figure  313 3a,b), compared to mock-inoculated plants (GLM, χ² = 0.0007 and χ² = 0.0409 for Arabidopsis and 314 Camelina, respectively) and correlated with the strong symptoms of infected plants.  (Figure 4a,b) for both plant species indicated that the three biological replicates per 325 condition clustered well together and that the different conditions (mock-inoculated or infected with 326 either virus) were well separated. Thus, the reads were of excellent quality and suited for a 327 transcriptome analysis. 328 For 8 selected Arabidopsis genes, the trends of gene deregulations detected in the transcriptome 329 data could be reproduced by an alternative analysis method, RT-qPCR (Supplementary Figure S1  330 RT-qPCR). All 8 genes followed the same trend using either method for CaMV-infected 331 Arabidopsis, and all except two (At_AOS and At_EDS5) for TuYV-infected Arabidopsis. The 332 discrepancies were probably due to the rather weak expression changes, which are sometimes 333 difficult to detect by RT-qPCR due to its intrinsic exponential amplification kinetics. 334 Quantification of viral RNA loads by counting viral reads normalized per million of total plant 335 reads in each sample revealed that CaMV pregenomic (pg)RNA and TuYV genomic (g)RNA (both 336 represented by forward reads; Supplementary Dataset S1) accumulated to comparable levels in each 337 of the three biological replicates, with the exception of one of the three TuYV-Arabidopsis 338 replicates which showed a lower number of normalized viral reads. The data confirmed further that 339 the mock-inoculated plants were not cross-contaminated. Note that, because TuYV gRNA is not 340 polyadenylated (unlike CaMV pgRNA) the poly(A) enrichment step of Illumina library preparation 341 protocol should have led to its depletion. This might explain the greater variation in relative 342 abundance of TuYV reads between biological replicates, compared to CaMV reads. Despite this 343 high variability, a lower virus load was observed in TuYV-infected Arabidopsis compared to 344 TuYV-infected Camelina plants (Supplementary Dataset S1). Notably, CaMV loads in Arabidopsis 345 were also lower than those in Camelina (ca. 1.5 times; Supplementary Dataset S1). This indicates 346 that despite drastic differences in disease symptoms between CaMV (severe symptoms) and TuYV 347 (mild symptoms) in both Arabidopsis and Camelina, Camelina appears to be more conductive for 348 replication of both viruses than Arabidopsis. 349  358 We determined the number of differentially expressed genes (DEGs) in infected hosts (Figure 4c-e). 359

CaMV modifies expression of far more genes than TuYV
Far more DEGs were detected in Camelina than in Arabidopsis. This was in part due to its 360 allohexaploid genome consisting of three Camelina genes. Also, the higher accumulation of both viruses in this host might contribute to the 365 higher counts. 366 In Arabidopsis, CaMV modified expression of ~11,800 genes significantly (P (adj) <0.05), 367 whereas TuYV modified expression of ~1,300 genes, corresponding to 43 % and 5 % of the total 368 genes, respectively ( Figure 4e). In CaMV-infected Camelina, we detected ~36,700 DEGs, and in 369 TuYV-infected Camelina ~9,400 DEGs, corresponding to 41 % and 11 % of all genes, respectively. 370 Thus, the impact of CaMV infection on gene deregulation was much more pronounced when 371 compared to TuYV infection, in accordance with the phenotype of infected plants ( Figure 1). The 372 lower numbers of DEGs for TuYV in both hosts could be at least partially due to its restriction to 373 phloem tissues, unlike CaMV which infects all cell types. 374 CaMV modified expression of ~40 % of the total genes independently of the host plant, whereas 375 the proportion of TuYV-induced DEGs was host-dependent and two times higher in infected 376 Camelina compared to Arabidopsis (11 % vs. 5 %). This is in line with the relative loads of viral 377 RNA (Supplementary Dataset S1), indicating that Camelina is more susceptible to TuYV infection 378 than Arabidopsis (3 times more TuYV RNA accumulation in Camelina compared to Arabidopsis), 379 while CaMV accumulates in both hosts at comparable levels (only 1.5-fold difference in average 380 viral RNA loads between Arabidopsis and Camelina). 381 956 DEGs, corresponding to 3.4 % of the genome, were common for both viruses in 382 Arabidopsis. The proportion of common DEGs rose to 7.5 % (~6,700 genes) in infected Camelina. 383 Since CaMV and TuYV are viruses with entirely different replication mechanisms, mediated 384 respectively by viral reverse transcriptase and viral RNA-dependent RNA polymerase, these 385 common host genes might be involved in general stress responses and/or are constituents of the core 386 defense mechanisms. A GO analysis indicated that this was true for Arabidopsis with GO terms 387 related to stress and transport in common for both infections, whereas for Camelina, ribosome and 388 replication-related genes were enriched (Supplementary Figure S2). 389 The proportions of up-and down-regulated genes were similar for a given virus in the two hosts 390 (

412
To identify the most prominent processes affected in aphid-infested CaMV and TuYV-infected 413 Arabidopsis and Camelina, we carried out a Gene Ontology (GO) analysis ( Figure 5). In general, 414 TuYV-induced GO changes were much less pronounced (considering the percentage of DEGs in 415 each category and the DEG counts) compared to CaMV, reflecting the low absolute numbers of 416 DEGs in TuYV-infected plants and the weaker impact of TuYV on plant phenotype. Remarkably, 417 in the Top 25 categories, only about 25 % of genes per GO were deregulated in TuYV-infected 418 Arabidopsis (Figure 5a), whereas this value increased to more than 50 % in TuYV-infected 419 Camelina (Figure 5c)

471
CaMV and TuYV infection downregulated photosynthesis-related genes in infested Arabidopsis 472 and Camelina ( Figure 6). Overall downregulation of photosynthesis-related genes was much more 473 pronounced in CaMV-infected than in TuYV-infected plants. This suggests that aphid preference for a plant is not only driven by visual aspects. 503

509
In line with the repression of photosynthesis, expression of many sucrose synthesis and 510 gluconeogenesis-related genes was reduced by CaMV infection (Figure 7a,b). The effect of CaMV 511 was stronger in Camelina than in Arabidopsis. In TuYV-infected Camelina, the amplitude of the 512 gene deregulation was smaller, compared to CaMV-infected Camelina, but the proportions of up-513 and down-regulated genes were comparable. In TuYV-infected Arabidopsis, expression changes 514 were even smaller than in TuYV-infected Camelina. For both TuYV-and CaMV-infected plants, 515 among the most down-regulated genes were those coding for key enzymes in sucrose synthesis, in 516 particular HCEF1 (AT3G54050) and FBP (AT1G43670). Interestingly, the sucrose phosphatase 517 SPP1 (AT1G51420) was strongly upregulated by CaMV infection, but downregulated in TuYV-518 infected plants. The most down-regulated gene in gluconeogenesis was the aldolase FBA5 519 (AT4G26530) and this in all four conditions tested. In line with the stronger suppression of 520 gluconeogenesis and sucrose synthesis-related genes by CaMV infection, many starch synthesis-521 related genes were also repressed by CaMV (but not TuYV) infection (Figure 7c). An exception 522 was When looking at global virus defense-related genes, the effects on their regulation were more 552 virus-specific than host-specific. In agreement with previous findings in CaMV-infected 553 Arabidopsis (Shivaprasad et al., 2008), many RNA silencing-related genes were found to be 554 upregulated by CaMV not only in Arabidopsis but also in Camelina (Figure 8a). Among them, most 555 notable are components of the 21 nt siRNA-directed gene silencing pathways such as Double-556 stranded RNA-binding protein 4 (DRB4), a partner of the antiviral Dicer-like protein 4 (DCL4) 557 generating 21 nt siRNAs, and siRNA-binding effector proteins Argonaute 1 (AGO1; AT1G48410), 558 AGO2 (AT1G31280) and AGO3 (AT1G31290). Notably, AGO2, also known to be involved in 559 defense against RNA viruses (Carbonell and Carrington, 2015), was up-regulated in TuYV-infected 560 Camelina ( Among components of other defense pathways (Figure 8b) Next, we looked at salicylic acid (SA) synthesis as this phytohormone is related to innate 606 immunity-based defense responses against non-viral and viral pathogens including CaMV (Zvereva  607  et

628
Next, we analyzed different metabolic pathways to determine if CaMV and TuYV infections 629 modulate other hormones and secondary metabolites in ways that are more favorable for their aphid 630 vector, and hence, for their own transmission. 631 We first looked at jasmonic acid (JA) and its derivatives because they are plant signaling 632 molecules related to plant defense against herbivorous insects, microbial pathogens and different 633 abiotic stresses (Figure 9a). We observed a strong virus-specific and host-independent effect for JA 634 synthesis genes. CaMV downregulated many genes in the JA pathway, while TuYV upregulated 635 some. Like for other pathways, the effect was stronger in infected Camelina than in Arabidopsis. 636 Deregulated genes were for example AOC1/3/4 (3 out of for 4 chloroplastic allene oxide cyclases, 637 involved in JA synthesis), AOS (AT5G42650, chloroplastic allene oxide synthase, involved in JA 638 synthesis) and LOX2 (AT3G45140, chloroplastic lipoxygenase required for wound-induced JA 639 accumulation in Arabidopsis). All these genes were slightly upregulated in TuYV, and strongly 640 repressed in CaMV-infected plants. This might imply that JA production is down in CaMV-infected 641 plants and stable or slightly induced in TuYV-infected plants. JA is generally thought to decrease 642 aphid growth and fecundity, so aphids on CaMV-infected plants might have greater fecundity. 643 However, infection of Arabidopsis with CaMV lowered fecundity (Figure 3a). JA-mediated 644 signaling pathways are also known to increase proteins and secondary metabolites, which act as 645 feeding deterrents (Howe and Jander, 2008). In this context, decreased JA production in CaMV-646 infected Arabidopsis could encourage longer/faster phloem sap ingestion, which we observed 647 indeed in our experiments (Figure 2a). Interestingly, phloem sap ingestion has been correlated with 648 increased CaMV acquisition (Palacios et al., 2002), which makes JA pathway a major candidate for 649 virus manipulation. 650 We also analyzed ethylene (ET) synthesis (Figure 9b)   CaMV-infected plants (Figure 2). 755 Next, we examined expression of genes known to be involved in plant responses and defenses 756 against insects (Figure 10b), as their modulation could influence virus-insect interactions and hence 757 transmission. General trends were suppression in CaMV-infected Camelina and activation in 758 CaMV-infected Arabidopsis and in TuYV-infected Camelina and Arabidopsis, resulting in both 759 host-specific and virus-specific responses. ESM1 (AT3G14210) was strongly downregulated in both 760 CaMV-infected hosts, but not in TuYV-infected hosts. Its gene product biases production of 761 glucosinolates, and its knockout mutant is more susceptible to herbivory by the caterpillar 762 Trichoplusia ni (Zhang et al., 2006). Arabidopsis. All in all, plant defense responses against insects did not follow a clear pattern. This 778 was probably due to the very divergent pathways and the heterogeneity of the plant insect response 779 genes. 780

Concluding remarks 781
In this work we analyzed the effect of CaMV and TuYV infection of M. persicae aphid-infested 782 Arabidopsis and Camelina on the plant hosts' transcriptomes as well as on the fecundity and 783 feeding behavior of their vector M. persicae. 784 Our results show that CaMV infection caused more severe effects on phenotype of both plant 785 species than did TuYV infection (Figure 1). The severity of symptoms correlated strongly with the 786 proportion of DEGs (41-43 % for CaMV, 5-11 % for TuYV, Figure 4e). CaMV infection affected 787 the same percentage of genes in both plant hosts, whereas TuYV infection deregulated 788 proportionally twice as many genes in Camelina than in Arabidopsis. Again, this correlated with 789 stronger visible symptoms on TuYV-infected Camelina in comparison with TuYV-infected 790 Arabidopsis. Aphid performance changes were more pronounced on CaMV-infected hosts, 791 whatever the plant species, compared to those caused by TuYV infection. In spite of more DEGs in 792 TuYV-infected Camelina than in TuYV-infected Arabidopsis, aphid behavior was slightly more 793 impacted on TuYV-infected Arabidopsis (Figure 2). This likely indicates modification of plant 794 metabolites that cannot be identified by transcriptome profiling. A metabolomic analysis of virus-795 infected leaves or phloem sap should provide complementary data on the aphid-plant-virus 796 interactions. 797 In this study, we did not probably also in virus-infected and infested plants could be minor. 802 The most pronounced effect of CaMV infection on plant hosts was a strong downregulation of 803 photosynthesis genes ( Figure 6) and carbohydrate metabolism-related genes (Figure 7). We 804 observed significant changes in many other pathways, including categories that are likely affecting 805 virus-vector interactions (i.e. defenses, silencing, hormones, secondary metabolites etc.). However, 806 the impact of these modifications on aphid fitness or feeding behavior was not easy to evaluate 807 since these parameters are likely under the control of several, often overlapping metabolic 808 pathways. Trying to correlate the effect of specific genes on aphids as reported in the literature with 809 our aphid behavior observations therefore often resulted in contrasting results. We offer the 810 following explanations. The very strong alterations in photosynthesis might have drowned 811 otherwise visible effects of DEGs previously found to be involved in plant-aphid interactions. 812 Another explanation is regulation by posttranscriptional and posttranslational modifications. While 813 transcriptome profiling is a powerful tool, it can display only changes of transcript levels. In many 814 cases, however, posttranslational modifications of proteins (such as phosphorylation, localization, 815 complex formation and many more) and even posttranscriptional RNA modifications (sequestering 816 of RNAs in p-bodies and others) will contribute to phenotype changes. Depending on the pathway, 817 the contribution of the transcriptome and posttranscriptome on cellular processes and beyond will 818 vary. This again indicates that complementary analyses such as metabolomics, proteomics etc. 819 might help to gain a more complete insight. 820 Nevertheless, we observed that virus infections have very distinct effects on the transcriptome of 821 host plants, and that, as expected, the non-phloem-limited virus (i.e. CaMV) has a significantly 822 stronger impact on plant hosts than the phloem-limited virus (i.e. TuYV). Overall, viral infection 823 with CaMV tends to have effects on metabolic pathways with strong potential implications for 824 insect-vector / plant-host interactions, while TuYV only weakly alters these pathways. For example, 825 the strong gene downregulations in the jasmonic acid, ethylene and glucosinolate biosynthetic 826 processes (Figure 9a-c) in CaMV-infected plants could be responsible for the observed alterations 827 of aphid feeding behavior and performance. Next steps could consist of functional validation of 828 some candidate genes identified in our study for their role in viral manipulation and consequently 829 potential impacts on viral transmission. 830

Author contributions
Supplementary Dataset S1 Plant mRNA-seq.xlsx 1148 Supplementary Dataset S2 Heatmaps DEGs List.xlsx 1149 Supplementary Sequence Information S1 on CaMV and TuYV.docx 1150 1151   Quality of RNA and sequence alignment data section "Therefore, TuYV accumulation as judged by our RNA-seq data might not be accurate and indeed, we found a difference between Western blot results and RNA-seq data (Supplementary Dataset S1). Concerning CaMV, its loads were lower in Arabidopsis than in Camelina (ca. 1.5 times; Supplementary Dataset S1), in line with the Western blot results (Figure 1d)." Legend to Figure 1 "c-d) Western blot analysis of TuYV CP coat protein (c) and CaMV coat protein P4 (d). On each lane, a total extract from a different plant was loaded. Ponceau staining of the small RuBisCO subunit is shown as a loading control." 2. The results section is very long and dense. I would suggest breaking it up into more sub-sections, with each heading specifying the main finding of that sub-section. This is especially the case for the heatmap analyses. It is difficult to get the main picture from this section or to be able to revisit certain pathways discussed without some kind of headings as a roadmap.
We shortened the text and divided the section on heatmaps in four subsections, each headed by its own subtitle: "Photosynthesis-related genes responsive to CaMV and TuYV" "Carbohydrate pathways genes responsive to CaMV and TuYV" "Virus defense-related genes responsive to CaMV and TuYV" "Insect defense-related genes responsive to CaMV and TuYV" We hope that the section is now easier to read.
3. The authors suggest that functional validation of candidate gene involvement in virus-induced phenotypes and effects on aphid behavior could be a future step. However, it is very hard to pick out what targets are most promising from this analysis because of the density of the text. The authors could provide a summary table relating behavior/fecundity effects to key changes recorded in the transcriptome analysis. This could highlight changes that could underlie the behavior/fecundity response and those that run counter to it, with relevant literature citations.
We agree with the reviewer and added a summary table as suggested ( Table 1 in the "Concluding remarks" section). In this table, we have listed some candidate genes which seem to us the most promising for functional analyses. These genes were selected according to potential behavioral/performance effects on aphids by referring to the existing literature.
Reviewer #2 (Comments for the Author): General Comments: The main question of this paper is to address if RNA-Seq can tell if plant specific or virus-specific responses to insect feeding with the same vector insect can be detected. There are 6 different conditions including TuYV in Arabidopsis and Camelina, CaMV in Arabidopsis and Camelina and no virus in Arabidopsis and Camelina. There is a lot of data to go through and the authors present the majority of their RNA-Seq data as heat maps of only the relevant conditions, pathways and genes of interest between each of the plants and viruses. There is also inclusion of the general RNA-Seq data points of concern including total numbers of reads, total numbers of upregulated vs downregulated genes, etc. The authors also tested a number of aphid specific conditions such as feeding activity and fecundity. This is a very thorough study which is original in the terms of the breadth of coverage and the number of conditions tested to compare to each other. This reviewer is astounded and excited about the authors undertaking this enormous project and the sheer amount of data that was produced.

Thank you.
Paper is moderately well written, there are a number of small things that could clarify and make the paper more readable. Due to the sheer amount of data presented, it is easy to get lost and distracted in the paper on avenues which can be difficult to follow. Difficult in the sense that it required movement not only through the paper, but also through the extensive amount of supplementary data as well.
Authors do not clarify the use of some terms used often in the results section and this could be much improved by simply putting the log2fold in parenthesis next to the transcript in question. For example: Line 481-"The most down-regulated gene in CaMV-infected Arabidopsis, AT3G27690, encoding a protein LHCb2.4, a component of the light harvesting complex, was also strongly repressed in Camelina infected by CaMV". In this example, the log2fold of the Arabidopsis gene could be put in the parenthesis next to that gene and the log2fold of the Camelina could also be put in parenthesis next to that reference. The author uses terms like "strongly downregulated", "strongly repressed", "strongly induced" and so on with no clear definition on what that means to them. Simply including the numbers in the writing when referencing the gene itself would clarify the writing substantially and not require the reader to constantly look back at the supplementary data which is exhausting and time consuming.
Considering that the authors do not talk about all of the changes in gene expression-of which there were many-adding the numbers to the ones that were deemed important enough to talk about in the writings will make this paper much more understandable as to what the author means by the use of these general and subjective terms.
We agree that for better visibility it is pertinent to add the log-fold changes to the expression statements directly into the text. Accordingly, we added the numbers to the cited genes.
In the methods, please clarify why the use of 18 day time points were utilized for both viruses for the placement of insects.  Figure 1 to show leaf purpling on TuYV-infected Arabidopsis and yellowing on TuYV-infected Camelina.
There is still some disconnect between the EPG data and the RNA-Seq. There is also some disconnect in the fecundity and the RNA-Seq as well. This appears most prominent in the discussion and when these topics are not discussed in the concluding remarks in relation to the RNA-Seq. Weak expression changes are difficult to measure with qPCR, due to its exponential amplification characteristics.
We selected both high expression and low expression genes to validate the results of bioinformatics analysis of the RNA-seq data. We did not specifically explore any genes with alternatively spliced transcripts.
Indeed, we did not analyze Camelina genes because we experienced difficulties with the multiple gene forms.
In the concluding remarks-Line 796-the authors state that an earlier study looked at only the insect feeding and noticed the response was much less then their current study. Given that the reviewer was expecting these conditions to be tested in this study as a further control, this reviewer feels that should be elaborated on to include any gene changes that were shared between the studies. Since it was in Arabidopsis, the Gene IDs should be the same and could be compared to look at it instead of just mentioning it offhand.  (265) suggesting that the contribution of aphid infestation to the transcriptome changes in healthy (and probably also in virus-infected and aphid-infested plants) is minor. Although it is difficult to directly compare their data with ours, we looked for Arabidopsis genes that were upregulated by aphid infestation alone but downregulated by aphid infestation plus virus infection. The rationale was that these genes might reflect viral effects to reduce the host plant's capability to recognize aphid infestation and establish defenses, thus favoring aphid infestation. For TuYV, only one gene that was upregulated by aphid infestation was downregulated by concomitant TuYVinfection (the transcription factor DREB26, AT1G21910). But 36 genes were downregulated by CaMV while upregulated by Myzus infestation alone (none inversely). GO analysis of these genes indicated an enrichment of genes related to 'response to chitin', 'response to salicylic acid', 'response to salt stress', 'response to wounding', 'hormone-mediated signaling pathway ', 'defense response to fungus', 'regulation of defense response' and 'signal transduction' (see Supplementary Table S3). This indicates that at least CaMV might dampen plant perception of aphid infestation and defenses against aphids, which might manifest itself in that aphids reach the phloem faster and feed longer on CaMV-infected plants. The fact that Myzus fecundity was lower on these plants, might be explained by the profound changes in other GOs, especially photosynthesis and carbohydrate metabolism, which probably reduce the nutritional value of CaMV-infected plants. Some of these genes could merit further exploration." Introduction contains all relevant background required to determine the importance of the study, suggest no changes.

Thank you.
References seem well ordered and formatted, suggest no changes.
Thank you.
Scientific method and experiments are well conducted, suggest addressing why Camelina was not confirmed via qRT-PCR. Also include the above mentioned comment about why 18 days was utilized as a time point.

Please see above.
Reviewer comments: Reviewer #1 (Comments for the Author): This is a technically sound and interesting paper that presents data on vector behavior/fecundity in relation to infected and healthy hosts, contrasting two very different viruses each infecting two related, but distinct host species. This is followed up with a transcriptomic analysis of a different set of infected and healthy hosts of each species x virus combination in the same stage of growth/disease progression as that used for behavior/fecundity experiments. I have just a few suggestions for improvement of the manuscript.
1. It would be nice to know why virus replication levels were only quantified through the transcriptome data, and not using an RT-qPCR assay that avoids the issue of TuYV not having poly A tails. Although differences were still found, it would be good if these were verified with a more standard diagnostic method for evaluating virus replication in the host.
As an alternative approach to assess virus accumulation we added to Figure 1  The methods section, the results sections "Plant Phenotype" and "Quality of RNA and sequence alignment data" and the legend to Figure 1 were accordingly adapted: Method section:

"Western blotting
Total protein extracts were prepared from leaves, separated by SDS-PAGE and transferred to nitrocellulose as described previously (Chesnais et al., 2021). Western blots were performed using antisera raised against CaMV P4 (Champagne et al., 2004) and TuYV CP (Bruyère et al., 1997). Secondary antibodies were horseradish peroxidase conjugates, and bound antibodies were revealed by enhanced chemiluminescence."

Plant phenotype section:
"Western blot analysis showed that TuYV accumulated, as previously reported (Claudel et al., 2018), to similar levels in Arabidopsis and Camelina. Thus there was no obvious link between TuYV accumulation and severity of symptoms, since despite comparable TuYV loads in Arabidopsis and Camelina, disease symptoms were stronger in Camelina than in Arabidopsis (Figure 1; compare the stunted phenotype of TuYV-infected Camelina with the weak phenotype of TuYV-infected Arabidopsis). CaMV loads were higher in Camelina than in Arabidopsis; whether this correlated with symptom expression, was difficult to access because of the severe phenotype in both hosts." Quality of RNA and sequence alignment data section: "Therefore, TuYV accumulation as judged by our RNA-seq data might not be accurate and indeed, we found a difference between Western blot results and RNA-seq data (Supplementary Dataset S1). Concerning CaMV, its loads were lower in Arabidopsis than in Camelina (ca. 1.5 times; Supplementary Dataset S1), in line with the Western blot results (Figure 1d)." Legend to Figure 1: "c-d) Western blot analysis of TuYV CP coat protein (c) and CaMV coat protein P4 (d). On each lane, a total extract from a different plant was loaded. Ponceau staining of the small RuBisCO subunit is shown as a loading control." 2. The results section is very long and dense. I would suggest breaking it up into more sub-sections, with each heading specifying the main finding of that sub-section. This is especially the case for the heatmap analyses. It is difficult to get the main picture from this section or to be able to revisit certain pathways discussed without some kind of headings as a roadmap.
We shortened the text and divided the section on heatmaps in four subsections, each headed by its own subtitle: "Photosynthesis-related genes responsive to CaMV and TuYV" "Carbohydrate pathways genes responsive to CaMV and TuYV" "Virus defense-related genes responsive to CaMV and TuYV" "Insect defense-related genes responsive to CaMV and TuYV" We hope that the section is now easier to read.
3. The authors suggest that functional validation of candidate gene involvement in virus-induced phenotypes and effects on aphid behavior could be a future step. However, it is very hard to pick out what targets are most promising from this analysis because of the density of the text. The authors could provide a summary table relating behavior/fecundity effects to key changes recorded in the transcriptome analysis. This could highlight changes that could underlie the behavior/fecundity response and those that run counter to it, with relevant literature citations.
We agree with the reviewer and added a summary table as suggested ( Table 1 in the "Concluding remarks" section). In this table, we have listed some candidate genes which seem to us the most promising for functional analyses. These genes were selected according to potential behavioral/performance effects on aphids by referring to the existing literature.
Reviewer #2 (Comments for the Author): General Comments: The main question of this paper is to address if RNA-Seq can tell if plant specific or virus-specific responses to insect feeding with the same vector insect can be detected. There are 6 different conditions including TuYV in Arabidopsis and Camelina, CaMV in Arabidopsis and Camelina and no virus in Arabidopsis and Camelina. There is a lot of data to go through and the authors present the majority of their RNA-Seq data as heat maps of only the relevant conditions, pathways and genes of interest between each of the plants and viruses. There is also inclusion of the general RNA-Seq data points of concern including total numbers of reads, total numbers of upregulated vs downregulated genes, etc. The authors also tested a number of aphid specific conditions such as feeding activity and fecundity. This is a very thorough study which is original in the terms of the breadth of coverage and the number of conditions tested to compare to each other. This reviewer is astounded and excited about the authors undertaking this enormous project and the sheer amount of data that was produced. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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