NbALY916 is involved in potato virus X P25‐triggered cell death in Nicotiana benthamiana

Abstract Systemic necrosis often occurs during viral infection of plants and is thought mainly to be the result of long‐term stress induced by viral infection. Potato virus X (PVX) encodes the P25 pathogenicity factor that triggers a necrotic reaction during PVX‐potato virus Ysynergistic coinfection. In this study, we discovered that NbALY916, a multifunctional nuclear protein, could interact with P25. When NbALY916 expression was reduced by tobacco rattle virus (TRV)‐based virus‐induced gene silencing, the accumulation of P25 was increased, which would be expected to cause more severe necrosis. However, silencing of NbALY916 reduced the extent of cell death caused by P25. Furthermore, we found that overexpression of NbALY916 increased the accumulation of H2O2 and triggered more extensive cell death when coexpressed with P25, even though accumulation of P25 was itself reduced by the increased expression of NbALY916. Furthermore, transient expression of P25 specifically induced the expression of NbALY916 mRNA, but not the mRNAs of three other ALYs in Nicotiana benthamiana. In addition, we showed that silencing of NbALY916 or transient overexpression of NbALY916 affected the infection of PVX in N. benthamiana. Our results reveal that NbALY916 has an antiviral role that, in the case of PVX, operates by inducing the accumulation of H2O2 and mediating the degradation of P25.

proteins and relocalize two of them from the nucleus to the cytoplasm (Uhrig et al., 2004). The two other ALY proteins repress the silencing suppressor activity of P19 by altering its localization from the cytoplasm into the nucleus (Canto et al., 2006).
Potato virus X (PVX), a positive-sense single-stranded RNA virus, belongs to the genus Potexvirus and its genome carries a block of three partially overlapping open reading frames termed the "triple gene block" (TGB) that encode proteins required for virus cell-to-cell movement, among which TGBp1 (termed P25) is a multifunctional protein that acts as a viral suppressor of RNA silencing (VSR) and participates in organization of the viral replication complex (VRC) (Chiu et al., 2010;Tilsner et al., 2012). Furthermore, P25 is a pathogenicity factor that triggers cell death in PVX-associated synergisms and also is a putative avirulence (avr) protein inducing plant hypersensitive response (HR)-like cell death in a threshold-dependent manner (Aguilar et al., 2015(Aguilar et al., , 2018. Gonzalez-Jara and colleagues found that the enhancement of pathogenicity associated with the synergistic interaction of PVX and plum pox virus (PPV) is not a consequence of more efficient PVX replication due to RNA silencing suppression by the PPV VSR, HC-Pro (Gonzalez-Jara et al., 2005), but was related to the accumulation level of the PVX P25 protein. In our experiments, we found that transient expression of P25 using a high concentration of agrobacteria triggered an HR-like response at 9 days postinfiltration (dpi) in Nicotiana benthamiana leaves, whereas treatment with the same concentration of agrobacteria but lacking the P25 expression plasmid did not induce an HR ( Figure S1).
The unfolded protein response (UPR) was shown to contribute to cell death induced by P25-associated PVX/PPV synergism (Aguilar et al., 2018), but Ye et al. found that agroexpression of PVX P25 was a much weaker inducer of UPR-related genes than PVX TGBp3 (Ye et al., 2011). Hence, it was hypothesized that P25 might trigger HR by other non-UPR-related processes.
To identify potential host proteins that interact with the PVX P25 or participate in P25-mediated HR, P25 fused at the C-terminus to green fluorescent protein (GFP) was transiently expressed in N. benthamiana leaves. Putative plant P25-interacting proteins were collected by coimmunoprecipitation (Co-IP) with GFP-binding magnetic beads and identified by mass spectrometry (MS) ( Figure S2). MS results revealed 98 plant proteins that potentially interacted with P25 (Table. S1). Among them was Hin19 (Q6JAC7), called NbALY916 (AM167906.1) in N. benthamiana, which was shown to be involved in Nep1 Mo (a Nep1-like protein from Magnaporthe oryzae)-triggered hypersensitive cell death (Teng et al., 2014).
These results indicated that P25 interacts with NbALY916 in the nucleus in BiFC assays. Co-IP was then performed to verify the interaction between NbALY916 and P25. In this assay, plasmids encoding NbALY916-GFP and P25 were transiently expressed in N. benthamiana, with free GFP being expressed as a noninteracting control protein, and using GFP-TRAP_M beads to collect GFP and GFP-tagged proteins from the infiltrated leaf extract. P25 was precipitated in the presence of NbALY916-GFP but not with free GFP or when expressed alone in the plant (Figure 1b).
It was reported that silencing of NbALY916 by tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) compromised the cell death triggered by Nep1 Mo in N. benthamiana (Teng et al., 2014).
To test whether NbALY916 is also involved in the P25-induced HR, we similarly used TRV VIGS to silence NbALY916 in N. benthamiana plants before infiltrating them with P25. At 10 dpi, the NbALY916- showed that the accumulation of H 2 O 2 and percentage of electrolyte leakage in P25-infiltrated patches in TRV:NbALY916-silenced leaves was less than that in P25-infiltrated patches in nonsilenced leaves.
At 9 dpi, we could observe cell death in P25-containing patches in TRV:00-treated leaves, but the cell death triggered by P25 was compromised in the TRV:NbALY916-treated leaves (Figure 2a, bottom panels). These results indicate that silencing NbALY916 inhibited cell death caused by P25 expression. We used RT-qPCR to detect the expression levels of NbrbohA and NbrbohB, which encode plant NADPH oxidases and are responsible for generating the H 2 O 2 involved in the HR (Yoshioka et al., 2003). In our experiments, the expression of NbrbohA and NbrbohB induced by P25 expression was significantly decreased in NbALY916-silenced leaves compared with TRV:00-treated leaves ( Figure 2c).
Western blotting was performed at 5 dpi to detect the accumulation of the P25 protein, showing that in TRV:NbALY916-treated leaves P25 accumulation was higher than in TRV:00-treated leaves.
Semiquantitative PCR results showed that the mRNA levels of P25 and TRV coat protein (CP) were not affected and NbALY916 was indeed silenced in TRV:NbALY916-treated leaves ( Figure 2d).
The increased level of P25 should cause more severe necrosis in unfused NbALY916 had the same effects ( Figure S4). Also, RT-qPCR analysis showed that the mRNA level of NbrbohA was up-regulated by expression of NbALY916-GFP, but not by free GFP (Figure 3c).
The mRNA levels of NbALY916 were increased at 3, 5, 7, and 9 dpi in patches infiltrated with P25 compared with control leaves ( Figure   S5a), but this was not the case with the other ALY mRNAs, NbALY617 and NbALY1693/NbALY615 ( Figure S5b,c).
We next investigated whether the extent of cell death was enhanced or not when P25 was coexpressed with NbALY916. We found at 5 dpi that coexpression of NbALY916-GFP and P25 trig- The main protein degradative routes in eukaryotes are the ubiquitin-26S proteasome system (UPS) and autophagy (Varshavsky, 2017). To elucidate which pathway was responsible for the degradation of the P25 protein when coexpressed with NbALY916, further coinfiltrations were performed with the addition of either MG132 (an inhibitor of the 26S proteasome pathway) or 3-methyladenine (3-MA, an inhibitor of autophagy) (Seglen and Gordon, 1982;Tanida et al., 2005). Western blotting showed that the accumulation of P25 when coexpressed with NbALY916 was not affected by treatment with MG132 or 3-MA ( Figure S6a). It was previously shown that autophagy could impact on the 26S proteasome's degradative route and remove excess or damaged proteasomes when the proteasome was inhibited by chemical or genetic deficiency (Marshall et al., 2015), which indicated that UPS and autophagy could complement one another to manage the recycling of nutrients and mitigate proteotoxic stress. Therefore, we decided to combine both 3-MA and MG132 treatments and then examine the accumulation of the P25 protein coexpressed with NbALY916-GFP or free GFP in the same leaves

HR-conferred resistance to viral infection restricts virus spread
and is accompanied by the induction of rapid cell death (Kombrink and Schmelzer, 2001). In some viral infections, the HR is initiated by Avr/R protein interactions. For example, the tomato R protein Sw-5b recognizes a conserved epitope in the tospovirus NSm protein and triggers the HR to confer broad-spectrum resistance (Zhu et al., 2017). Cell death or systemic necrosis is also a feature of the plant response in many compatible viral infections. It is reported that the systemic necrosis caused by PVX-associated synergisms is  Table S2. Bars represent the SEM from three biological repeats. A two-sample unequal variance directional t test was used to test the significance of the difference (**p < .01). (d) Western blotting and reverse transcription PCR detection of the P25 protein and P25, TRV-CP and NbALY916 mRNAs in NbALY916-silenced and nonsilenced (TRV:00) leaves. The level of P25 protein accumulation was calculated relative to that of RuBisCO and the transcription levels of P25, TRV CP and NbALY916 were normalized relative to UBC (Ubiquitin C). The relative protein and mRNA levels were calculated using ImageJ a threshold-dependent immune response induced by P25 (Aguilar et al., 2015). Silencing of the host genes SGT1 and RAR1, or overexpression of the endoplasmic reticulum luminal binding protein (BiP), alleviated the HR (Aguilar et al., 2015(Aguilar et al., , 2018. In our study, we identified a new host nuclear protein NbALY916 that is involved in the P25-triggered HR. Furthermore, we revealed that transient over- Leaf discs were excised and assayed for electrolyte leakage at 5 dpi. Bars represent the SEM from three biological repeats, each consisting of six plants. A two-sample unequal variance directional t test was used to test the significance of the difference (**p < .01). (c) The transcript levels of NbrbohA and NbrbohB after transient expression of NbALY916-GFP or free GFP. Bars represent the SEM from three biological repeats. A two-sample unequal variance directional t test was used to test the significance of the difference (*p < .05). (d) Visible necrosis (left panel) and H 2 O 2 accumulation (right panel, DAB stained) after transient expression of combinations of P25, NbALY916-GFP, empty vector (EV), and free GFP. The expression of red fluorescent protein (RFP) was used as the reference of heterogeneous expression. (e) Leaf discs were excised and assayed for electrolyte leakage at 5 dpi. Bars represent the SEM from three biological repeats, each consisting of six plants. Error bars show SD and the graph represents the combined data from three independent replicates. Letters on the graph denote statistically significant differences (analysis of variance, p ≤ .05). (f) Western blotting detection of P25, NbALY916, and RFP in infiltrated leaves mentioned in (c). The value of P25 protein accumulation was normalized to RuBisCO. The indicated bands of NbALY916-GFP were used to calculate the protein accumulation levels by ImageJ. (g) P25 coexpressed with ALY916-GFP or GFP in the same leaf for 48 hr, followed by 10 mM 3-methyladenine (3-MA) and 100 μM MG132 for 4 hr. Western blotting detection of P25 and NbALY916 expressed with (+; 4 hr postinfiltration) or without (−) the addition of a mixture of 10 mM 3-MA and 100 µM MG132. The P25 protein accumulation was normalized to RuBisCO and the relative protein levels were calculated in relation to the 1% dimethyl sulphoxide-only treatment. The relative protein levels were calculated by ImageJ. The asterisks indicate bands of NbALY916-GFP in western blots (c) and (d) PVX coat protein (CP) and viral RNA accumulation in inoculated (at 3 dpi) and systemically infected (at 6 dpi) leaves of TRV:00-and TRV:NbALY916-treated plants, detected by western blotting and northern blotting. The CP and viral RNA accumulation were normalized to RuBisCO and rRNA, respectively, and the relative levels were calculated in relation to TRV:00 treatment. The relative protein and RNA levels (using all viral RNA from PVX and rRNA) were calculated using ImageJ. (e) Agroinfiltrated PVX (OD 600 = 0.001) with NbALY916-GFP (OD 600 = 0.1) or GFP (OD 600 = 0.1) on the same leaf. (F) PVX CP and NbALY916-GFP accumulation in the same leaf (at 3 dpi) detected by western blotting. The CP and NbALY916-GFP accumulation were detected using anti-PVX CP and anti-GFP antibodies, respectively, and the relative levels were calculated in relation to coexpressed PVX and GFP using ImageJ. The asterisks indicating bands of NbALY916-GFP in western blots NbALY916 inhibited the accumulation of H 2 O 2 that normally occurs after treatment with the fungal Nep1 Mo cell death elicitor (Teng et al., 2014). Our observation that NbALY916 is also involved in the turnover of P25 by an as yet undiscovered mechanism suggests there could be a common pathway for NbALY916 interaction with pathogenicity elicitors from various plant pathogens as part of basal HRassociated plant defence.

ACK N OWLED G M ENTS
This work was financially supported by the Natural Science

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.