A naturally occurring PVY^NTN strain isolated from infected potato plants grown in a commercial field in Washington State was used. The virus was maintained on N. benthamiana plants. The complete RNA genome was obtained as cDNA by RT-PCR using six overlapping primer pairs (PVY1FP/PVY1RP, PVY2FP/PVY2RP, PVY3FP/PVY3RP, PVY4FP/PVY4RP, PVY5FP/PVY5RP, PVY6FP/PVY6RP) ( Supplementary Table 1 ). Overlapping amplicons were separately cloned into pJET1.2 vector and sequenced and the complete genome of PVY^NTN was deposited (GenBank accession no. ON866943).

To generate the entry clone, pDONR201-NIa-pro, the coding sequence of NIa-pro of PVY^NTN was PCR amplified using a primer pair NIaProFP/NIaProRP ( Supplementary Table 1 ), and was cloned into the entry vector, pDONR201. To generate pDONR201-IAAS construct, the full-length coding sequence of IAAS (GenBank accession no. ON866944) was PCR-amplified from the cDNA of N. benthamiana leaves by using the specific primer pair (IAASFP/IAASRP) and cloned into the pDONR201 vector. The pGBKT7-NIa-pro and pSITE-nEYFP-N1:NIa-pro constructs were generated by mobilizing the NIa-pro from pDONR201-NIa-pro to the pGBKT7-GW and pSITE-nEYFP-N1 vector, respectively. This in vitro recombination-mediated mobilization from an entry clone to the destination vector was performed by using Gateway™ LR Clonase™ enzyme mix (Thermo Fisher Scientific) by following the manufacturer’s instructions. Similarly, pGADT7-IAAS, pSITE-nEYFP-C1:IAAS, and pEARLEY103-IAAS constructs were generated by mobilizing the IAAS coding sequence from pDONR201-IAAS to pGADT7-GW, pSITE-nEYFP-C1, and pEARLEY103-GW vectors, respectively. To generate the pTRV-IAAS-silencing construct, IAAS-fragment (346 bp) was amplified using the primer pair SiIAASFP/SiIAASRP and cloned in antisense orientation in the pTRV2-GW vector. Similarly, to generate the pTRV-NbPDS-silencing construct, the fragment of N. benthamiana Phytoene desaturase (NbPDS) (491 bp) was amplified from the cDNA of N. benthamiana leaves by using the specific primer pair SiPDSFP/SiPDSRP ( Supplementary Table 1 ) and cloned in antisense orientation in pTRV2-GW vector. To generate the pGR106-IAAS expression construct, the full-length coding sequence of IAAS was amplified with the primer pair PExIAASFP/PExIAASRP, cloned into pJET.12 and subcloned in Cla1 and Sal1 restriction site of the pGR106 vector. Sequences of all the primers used in this study are listed in Supplementary Table 1 .

The pGBKT7-NIa-pro construct containing the coding sequence of NIa-pro of PVY^NTN in-frame with the yeast GAL4 binding domain protein was used as a bait construct for the Y2H library screening. The mRNA was isolated from leaves of the N. benthamiana plants using Oligotex mRNA Mini Kit (Qiagen) and by following the manufacturer’s instructions. The N. benthamiana Y2H cDNA library was constructed using mRNA isolated from the leaves of the N. benthamiana plants and Matchmaker^® Gold Yeast Two-Hybrid System kit (Takara). Construction of N. benthamiana Y2H cDNA library and screening were performed as illustrated in the Matchmaker Gold Yeast Two-Hybrid System User Manual (Takara). The N. benthamiana Y2H library was screened using pGBKT7–NIa-pro as the bait in Saccharomyces cerevisiae strain Y2HGold. Yeast colonies containing potential interacting partners of NIa-pro were selected based on the ability of the co-transformed cells to grow on synthetic media containing aureobasidin and lacking Trp and Leu. Subsequently, those yeast cells were screened with high stringency and were selected based on their ability to grow on synthetic media lacking Trp, Leu, and His and containing 1mM 3-amino-1,2,4-triazole. The prey plasmids present in the yeast colonies that were able to grow on synthetic media lacking Trp, Leu, and His and containing 3-amino-1,2,4-triazole were rescued and sequenced.

Y2H assays were performed by co-transforming S. cerevisiae strain Y2HGold cells with plasmid pGBKT7-NIa-pro plus pGADT7-IAAS. A pGBKT7-P53 and pGADT7-TAg plasmid combination was used as positive control, while a pGBKT7 and pGADT7 vector combination was used as the negative control. The interaction was tested by growing the yeast co-transformants on synthetic screening media lacking Leu, Trp, and His and containing 1 mM 3-amino-1,2,4-triazole and X-α-Gal at 30°C for 4 days (Gnanasekaran et al., 2019; Gnanasekaran et al., 2023).

BiFC assays were performed as described previously (Gnanasekaran et al., 2019). Leaves of three-week old N. benthamiana plants were co-infiltrated with Agrobacterium tumefaciens strain GV3101 cells containing pSITE-nEYFP-N1: NIa-pro and pSITE-nEYFP-C1: IAAS. Similarly, Agrobacterium cells containing pSITE-nEYFP-N1 and pSITE-nEYFP-C1, pSITE-nEYFP-N1: NIa-pro and pSITE-nEYFP-C1, pSITE-nEYFP-N1 and pSITE-nEYFP-C1: IAAS negative combinations were infiltrated into leaves of N. benthamiana plants. Forty eight hours post-infiltration, the abaxial epidermal cells of the infiltrated N. benthamiana leaves were examined using a Leica TCS SP8 X confocal laser-scanning microscope using 20X objective lens, and the LAS X software (Leica Microsystems, Wetzlar, Germany). The nuclei of the epidermal cells of the infiltrated leaves were stained with 4, 6-diamidino-2-phenylindole (DAPI) (Gnanasekaran et al., 2019). For detection of DAPI fluorescence, 405 nm blue diode was used for excitation and the emitted light was collected between 435 to 485 nm. For detecting reconstituted yellow fluorescent protein (YFP), 514 nm excitation was used and the emitted light was collected between 530 nm to 570 nm.

Subcellular localization of the NIa-pro and N. benthamiana IAAS protein was performed as described previously (Bhattacharyya et al., 2015). The leaves of three-week old N. benthamiana plants were infiltrated with A. tumefaciens strain GV3101 cells containing localization constructs (Bhattacharyya et al., 2015) namely, pCAMBIA1302 vector, pCAMBIA1302-NIa-pro, pEARLEY103-IAAS. The nuclei of the epidermal cells were stained with DAPI. After 48 hours post-infiltration, the abaxial epidermal cells of the infiltrated N. benthamiana leaves were examined using a Leica TCS SP8 X confocal laser-scanning microscope, and the LAS X software (Leica Microsystems, Wetzlar, Germany). For detection of DAPI fluorescence, 405 nm blue diode was used for excitation and the emitted light was collected between 435 and 485 nm. For green fluorescent protein (GFP) fluorescence, 488 nm excitation was used and the emitted light was collected between 515 nm to 545 nm.

Transient silencing was carried out using TRV-based gene silencing vectors. pTRV1 (CD3-1039) and pTRV2-GW (CD3-1041) vectors (Liu et al., 2002) were obtained from the Arabidopsis Biological Resource Center, OH, United States. pTRV1, pTRV2, pTRV2-IAAS, and pTRV2-NbPDS silencing constructs were transformed into A. tumefaciens strain EHA105. To achieve silencing of endogenous IAAS, two fully expanded top leaves of three-week old N. benthamiana plants were co-infiltrated with Agrobacterium cells carrying pTRV1 and pTRV2-IAAS. Plants with co-infiltrated Agrobacterium carrying either pTRV1 and pTRV2-NbPDS, or pTRV1 and pTRV2 combination served as positive and negative controls, respectively. The infiltrated plants were grown at 22°C, 70% relative humidity, and in 16h of light/8h of dark photoperiod with light intensity of 100 µmol/m²/s.

A modified potato virus X (PVX) vector, pGR106 (Lu et al., 2003; Gupta et al., 2022) was used for transient overexpression of IAAS in N. benthamiana plants. The pGR106 vector and pGR106-IAAS overexpression construct were mobilized into A. tumefaciens strain GV3101 containing helper plasmid, pJIC SA_Rep (Gupta et al., 2022). Overexpression of IAAS was accomplished by infiltrating the lower leaves of three week-old N. benthamiana plants with Agrobacterium cells carrying pGR106-IAAS. Plants infiltrated with Agrobacterium carrying pGR106 vector served as the control. The infiltrated plants were grown at 22°C, 70% relative humidity, and in 16h of light/8h of dark photoperiod with light intensity of 100 µmol/m²/s.

For PVY inoculation, leaves of the N. tabacum plants previously infected with PVY^NTN were used as source of inoculum. One gram of infected tobacco leaves was ground in 10 ml of 100 mM sodium phosphate buffer (pH 7.2) containing 0.4% (v/v) β-mercaptoethanol, and 1:10 (wt/vol) of carborundum (Manasseh et al., 2022). The sap extracted from PVY-infected leaf tissue was applied by manually rubbing the inoculum on the adaxial side of the leaves of N. benthamiana plants. While sap of healthy tobacco plants was used for mock-inoculation. PVY symptom severity were visually scored on a scale of 0-4 (0=no symptoms, 1= mild leaf curling and/or mild mosaic, 2= severe leaf curling and/or typical chlorosis, 3=mild necrosis and/or stunted growth, 4=severe necrosis). Symptom severity graphs were plotted using the score recorded from atleast 9 plants for each treatment and by using the sigma plot software.

The relative amount of the viral load present in the systemically infected top leaves of the infected plants was analyzed by Double-antibody Sandwich (DAS) Enzyme-Linked Immunosorbent Assay (ELISA). DAS-ELISA was performed using the ELISA reagent set (SRA 20001, Agdia, IN, USA). One gram of N. benthamiana leaf sample was ground in general extract buffer (GEB) (Agdia, Inc.). Leaf sap (100 µl) was used for measuring the viral titer using DAS-ELISA, with two technical replicates and three biological replicates for each treatment (Manasseh et al., 2022).

For each treatment, top two leaves from atleast three N. benthamiana plants were collected. Total RNAs from the collected N. benthamiana leaf tissues were extracted using RNeasy Plant Mini Kit (Qiagen, St. Louis, MO, USA) and were treated with DNase I (Sigma, St. Louis, MO, USA). The cDNA synthesis was carried out using random hexamers and MLV reverse transcriptase (Fermentas, USA). The RT-qPCR analysis was carried out as described previously (Zhai et al., 2019) with slight modifications. Protein phosphatase 2A (PP2A) was used as an internal reference gene (Cassan-Wang et al., 2012). RT-qPCR analysis performed in this study was carried out with three biological and three technical replicates. Statistical significance was determined by one-way ANOVA. Sequences of the primers used in this study are listed in Supplementary Table 1 .

Leaves of three- to four-week-old N. benthamiana plants were co-infiltrated with Agrobacterium cells carrying constructs that express either IAAS-GFP and N-terminal of YFP (YFP-N1) or IAAS-GFP and YFP-N1-NIa-pro. After 6 dpi, total protein from the infiltrated leaves was extracted in protein extraction buffer containing 1X phosphate buffer (pH 7.4), 5 mM β-mercaptoethanol, 1 mM Phenylmethylsulfonyl fluoride, and plant protease inhibitor cocktail (P9599, Sigma-Aldrich, USA). Equal amounts of total protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting assay using an anti-GST-HRP antibody (sc-9996 HRP, Santa Cruz Biotechnology, TX, USA).

N. benthamiana plants that were mechanically inoculated with sap containing PVY^NTN developed mild leaf curl symptoms at around 9 days post-inoculation (dpi) on systemically infected top leaves. Later at 15 dpi, plants showed severe chlorotic mosaic, leaf curling affecting the leaf lamina, and stunted growth, while the mock-inoculated N. benthamiana plants remained symptomless ( Figures 1A, B ). Similarly, N. tabacum plants inoculated with PVY^NTN, beginning at 15 dpi, showed severe veinal chlorosis affecting the mid-veins and later developed severe chlorotic mosaic at 20 dpi, while the mock-inoculated N. tabacum plants remained symptomless ( Figure S1 ). These observations suggested that PVY^NTN strain used in this study could induces a recognizable chlorotic mosaic symptoms on the leaves of both N. benthamiana and N. tabacum plants. The complete genome of PVY^NTN isolate used in this study was deposited in GenBank database (GenBank accession no. ON866943). The sequence showed a nucleotide identity of 99.79% with PVY^NTN isolate HR1 (GenBank accession no. FJ204166.1).

To explore the role of PVY-encoded NIa-pro protein during host-virus interaction, Y2H library was screened by using pGBKT7-NIa-pro as bait and N. benthamiana Y2H cDNA library. The N. benthamiana IAAS was found to be one of the potential host-interacting partners of NIa-pro protein. The interaction between the NIa-pro protein and N. benthamiana full-length IAAS protein was confirmed by Y2H assay. Yeast transformants carrying BD-NIa-pro and AD-IAAS plasmids were able to grow on selection plates deficient in Leu, Trp, and His and supplemented with 1 mM 3-amino-1,2,4-triazole and X-α-Gal ( Figure 2A ). The yeast transformants carrying positive control plasmids (AD-T_Ag and BD-P₅₃) grew on the selection plates, while yeast transformants with negative control plasmids (AD and BD, AD-IAAS and BD or AD and BD-NIa-pro) were unable to grow ( Figure 2A ). These results confirmed the interaction of NIa-pro protein with the IAAS protein in yeast cells.

Further, BiFC assays were carried out in N. benthamiana plants using the BiFC vectors, pSITE-nEYFP-N1 and pSITE-nEYFP-C1. The nuclei of the epidermal cells were stained using DAPI. YFP fluorescence was observed in the epidermal cells of the leaves co-infiltrated with Agrobacterium cells containing pSITE-nEYFP-N1:NIa-pro and pSITE-nEYFP-C1:IAAS constructs ( Figure 2B ; Figure S2 ). This YFP signal was detected predominantly in the cytoplasm and some in the nucleus and/or perinuclear regions. However, no YFP signal was observed in the epidermal cells of the leaves co-infiltrated with pSITE-nEYFP-N1 and pSITE-nEYFP-C1, pSITE-nEYFP-N1:NIa-pro and pSITE-nEYFP-C1, or pSITE-nEYFP-N1 and pSITE-nEYFP-C1:IAAS combinations ( Figure 2B ). These results demonstrate that NIa-pro protein interacts with IAAS protein.

The results of our BiFC experiment demonstrated that NIa-pro interacts with IAAS predominantly in the cytosol ( Figure 2B ). NIa-pro protein of PVY and TuMV, was shown to localize to the nucleus and/or vacuoles (Bak et al., 2017). To better understand the role of NIa-pro protein in the host-virus interaction, we performed subcellular localization assays. NIa-pro-GFP fluorescence was observed at the nuclei (co-localized with signal from DAPI), cytosol, and nuclear inclusion ( Figure S3 ). IAAS-GFP fluorescence also was observed in the cytosol, as well as the perinuclear and nuclear region, whereas fluorescence of the free GFP (control) was observed throughout the whole cell ( Figure S3 ), suggesting that both NIa-pro and IAAS independently localize to the cytosol and to some degree to the nucleus and/or perinuclear regions.

Silencing of the IAAS gene that encodes the IAAS protein was achieved in N. benthamiana plants by infiltrating Agrobacterium cells containing the pTRV-IAAS silencing construct, while the pTRV vector infiltrated plants served as the control for silencing experiments. The pTRV-NbPDS construct infiltrated N. benthamiana plants served as phenotypic control for gene silencing and showed photobleaching at10 dpi; ( Figure S4A ). The pTRV-IAAS-infiltrated N. benthamiana plants showed around 5 cm increased height compared to the pTRV- infiltrated plants ( Figure S4B ). IAAS transcript levels in the upper leaves of pTRV-IAAS or pTRV- infiltrated plants at 10 dpi were measured by semiquantitative RT-PCR. Silencing of the IAAS gene was reflective of the reduction in IAAS mRNA level in pTRV-IAAS infiltrated plants compared to pTRV- plants ( Figure S5 ).

To investigate the role of IAAS protein in PVY pathogenesis, IAAS-silenced plants and the pTRV-control N. benthamiana plants were inoculated with PVY^NTN isolate. PVY-inoculated pTRV control plants displayed typical symptoms such as leaf curling, severe chlorotic mosaic, and stunted growth, while PVY-inoculated IAAS silenced plants showed leaf curling and chlorotic mosaic but not stunted growth at 15 dpi ( Figure 3A ). PVY-infected pTRV control plants showed PVY symptoms at around 9 dpi, whereas PVY infected IAAS-silenced had showed delayed symptoms at around 11 dpi ( Figure 3B ). The accumulation of PVY virus-particle and viral RNA were assessed by DAS-ELISA and RT-qPCR, respectively. At 15 dpi, the accumulation of PVY in the upper leaves of PVY-infected IAAS silenced plants was found to be reduced compared to that in PVY-infected pTRV-control plants ( Figure 3C ). Similarly, IAAS silenced plants showed decreased PVY RNA level compared to that of pTRV-control plants ( Figure 3D ). Thus, silencing of IAAS reduced the susceptibility of N. benthamiana plants to PVY. Taken together, these results suggested that silencing of IAAS gene probably increases the level of free auxin and contributes to both promoting plant growth and reduced susceptibility of PVY in the N. benthamiana plants.

Transient overexpression of IAAS protein was accomplished in N. benthamiana plants using the pGR106-IAAS construct, while pGR106-infiltrated plants served as control. To determine the role of IAAS protein during PVY infection, we first analyzed plants infiltrated with pGR106-IAAS or pGR106-vectors without PVY challenge. The pGR106 vector, being a PVX-based plant expression vector, the mock-treated pGR106-IAAS- and pGR106-infiltrated plants showed mild symptoms of PVX. IAAS transcripts levels in upper leaves of pGR106-IAAS or pGR106-control infiltrated plants at 7 dpi were measured by semiquantitative RT-PCR. Transient overexpression of the IAAS gene was confirmed as an increased accumulation of IAAS mRNA in the pGR106-IAAS infiltrated plants as compared to pGR106-control plants ( Figure S5 ).

Later, to study the role of IAAS protein during the PVY pathogenesis, we inoculated IAAS-overexpressing plants and the pGR106-control N. benthamiana plants with PVY^NTN isolate. The PVY-mediated symptom induction was found to be enhanced in pGR106-IAAS-infiltrated N. benthamiana plants compared to pGR106-infiltrated plants ( Figure 4A ). The pGR106-IAAS-infiltrated N. benthamiana plants showed necrotic spots on leaves around 13 dpi ( Figure 4A ; Figure S6 ). PVY-infected IAAS overexpressing plants showed exacerbated PVY symptoms compared to that of pGR106-infiltrated control plants ( Figures 4A, B ) during later stage of infection (after 13 dpi). The accumulation of PVY virus particles and viral RNA was accessed by DAS-ELISA and RT-qPCR, respectively. At 15 dpi, the accumulation of PVY virus particles in the upper leaves of PVY-infected pGR106-IAAS-infiltrated plants was found to be significantly higher than the PVY-infected pGR106-infiltrated plants ( Figure 4C ). Similarly, IAAS overexpressing plants showed significantly higher PVY RNA level compared to that of pGR106-control plants ( Figure 4D ). These results indicate that overexpression of IAAS increases the susceptibility for PVY in N. benthamiana plants.

PVY NIa-pro interacts with IAAS protein and modulates PVY-induced symptoms and virus accumulation in N. benthamiana plants. To further study the possible importance of NIa-pro-IAAS interaction, we investigated the expression of auxin-responsive genes during PVY infection. For this purpose, N. benthamiana plants were inoculated with PVY and were tested for the relative expression of selected auxin-responsive genes, Auxin response factor 1 (ARF1), Auxin response factor 3 (ARF3), and small auxin upregulated RNA 3 (SAUR3) at 24 hpi, 48 hpi, and 7 dpi by RT-qPCR in the systemically infected leaves. Results showed that expression of ARF1, ARF3, and SAUR3 were not significantly altered at 24 hpi and 48 hpi in the PVY-infected N. benthamiana plants compared to mock-inoculated plants ( Figure S7 ). However, at 7 dpi, expression of all the tested auxin-responsive genes was found to be significantly downregulated in the PVY-infected N. benthamiana plants compared to mock-inoculated plants ( Figure 5 ).

This observation led us to the hypothesis that during PVY infection its NIa-pro protein interacts with and stabilizes IAAS protein, which would lead to increased conjugated IAA levels and subsequently downregulation of the auxin-responsive gene expression. To test this hypothesis, leaves of N. benthamiana plants were co-infiltrated with Agrobacterium cells carrying constructs that expresses either IAAS-GFP and N-terminal of YFP (YFP-N1) or IAAS-GFP and YFP-N1-NIa-pro. After six dpi, the level of IAAS-GFP was determined by Western blotting using anti-GFP antibody. At six dpi, IAAS protein accumulation was found to higher in the leaves infiltrated with IAAS-GFP and YFP-N1-NIa-pro expressing constructs compared to those infiltrated with IAAS-GFP and YFP-N1. Taken together, these results suggest that NIa-pro protein interacts with and stabilizes IAAS protein. This probably leads to an increased conjugated IAA level and contributes to downregulation of auxin-responsive genes.