Enhanced virus infection in Nicotiana benthamiana transiently overexpressing  MDP92 , encoding a tobacco MYB transcription factor

doi:10.21203/rs.3.rs-5087338/v1

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Enhanced virus infection in Nicotiana benthamiana transiently overexpressing MDP92 , encoding a tobacco MYB transcription factor

https://doi.org/10.21203/rs.3.rs-5087338/v1

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Transcription factors including MYB proteins are involved in plant resistance to viruses. However, the information on the role and function of MYB proteins during plant virus infection is limited. In this study, we found the trend that the expression of a tobacco MYB gene (MDP92) is temporarily be downregulated in tobacco cultivars with and without the virus resistance gene N during tobacco mosaic virus infection. To test a possible involvement of MDP92 in the regulation of virus infection, we investigated the effect of the transient overexpression of the MDP92 coding sequence alone or in combination with the N genomic sequence on infection with GFP-encoding tomato mosaic virus in Nicotiana benthamiana. Overexpression of MDP92 promoted virus intercellular movement in leaves in the presence of N and enhanced virus accumulation in leaves and protoplasts in the absence of N. Gene expression analysis of four selected resistance-related genes (NbPR1a, NbPR4, NbHin1, and NbHsr203j) showed that compared to control leaves, only NbHsr203j expression was significantly downregulated in MDP92-overexpressing leaves with N, and the expression of NbPR4 and NbHin1 was significantly upregulated in MDP92-overexpressing leaves without N. In contrast, NbPR1a expression was not affected by MDP92 overexpression. Transient overexpression of MDP92 without N also resulted in enhanced accumulation of potato virus X with upregulation of NbPR4 and NbHin1 expression during early virus infection. Based on these results, we discuss the role of the transcription factor MDP92 during virus infection.

MYB

virus resistance

N gene

TMV

ToMV

PVX

tobacco

Nicotiana benthamiana

Plant resistance to pathogens is primarily controlled by two defense mechanisms. The first mechanism involves the interaction between plasma membrane-associated receptors and pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity or basal resistance (Ngou et al. 2024). In the case of virus infection, virus-derived nucleic acids are shown to be PAMPs that induce RNA silencing and trigger callose deposition at plasmodesmata (Miyashita et al. 2016; Huang et al. 2023b). In the second mechanism, nucleotide-binding leucine-rich repeat receptors (NLRs) recognize pathogen-derived avirulence factors (also known as effectors or elicitors) to induce effector-triggered immunity or true resistance, which is commonly known as hypersensitive reaction (HR) associated with cell death (Huang et al. 2023a). These two defense mechanisms are positively or negatively regulated by a variety of transcription factor families, exemplified by the WRKY, AP2/ERF, bHLH, bZIP, NAC, and myeloblastosis (MYB) families (Meraj et al. 2020; Tsuda and Somssich 2015). However, due to the diversity of transcription factor families and their members and the complexity of transcriptional networks, the roles and functions of individual transcription factors in plant defense, especially against viruses, are only partially understood.

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are positive single-stranded viruses of the genus Tobamovirus. These tobamoviruses infect susceptible host plants such as tobacco (Nicotiana tabacum) and N. benthamiana systemically, causing mosaic and wilt symptoms (Okada 1999; Scholthof et al. 2011). In contrast, tobacco cultivars carrying the N resistance gene (NN tobacco), which encodes a Toll/interleukin-1 receptor (TIR) type NLR, induce HR to tobamovirus infection (Whitham et al. 1994). N protein recognizes the helicase domain of the TMV replicase as an elicitor (Erickson et al. 1999). Transcriptome analysis of TMV-infected, susceptible tobacco lacking the N gene (nn tobacco) or resistant NN tobacco has revealed significant alterations in the expression levels of genes involved in defense metabolism, plant-pathogen interaction, phytohormone signaling, and RNA silencing, as well as those encoding transcription factors (Golem and Culver 2003; Sheng et al. 2019). Transcription factors in the WRKY and AP2/ERF families are shown to be transcriptionally upregulated during N-mediated resistance induction in tobacco, which is thought to result in the modulated expression of defense-related genes (Gao et al. 2014; Kim and Zhang 2004; Menke et al. 2005; Nishiuchi et al. 2002; Ogata et al. 2012, 2015, 2022). Similar to these WRKY and AP2/ERF transcription factors, the NtMYB1 gene encoding a MYB transcription factor is upregulated in NN tobacco during TMV infection in association with the activation of pathogenesis-related genes including PR-1a (Yang and Klessig 1996). Silencing of the N. benthamiana homologs of an NtMYB1 gene (NbMYB1) as well as WRKY genes (NbWRKY1, NbWRKY2, and NbWRKY3) compromises TMV resistance in transgenic N. benthamiana expressing N (Liu et al. 2004). Another MYB-like gene in N. benthamiana, NbMYB4L, is induced by TMV infection and its increased expression is correlated with increased resistance to the virus (Zhu et al. 2022). In addition, NbAL7 of N. benthamiana has recently been identified as a novel transcriptional repressor that directly interacts with the N protein and positively regulates N-mediated resistance (Zhang et al. 2023). Thus, studies of transcription factors in Nicotiana species have focused primarily on positive regulators of virus resistance.

Transient production of the TMV elicitor domain (termed p50) alone and that in combination with the N protein can induce HR-like cell death in NN tobacco and nn tobacco, respectively (Abbink et al. 2001; Erickson et al. 1999; Gao et al. 2007; Mestre and Baulcombe 2006; Taku et al. 2018). In addition, transient co-production of p50 and the N protein in N. benthamiana reduces the infection with TMV, ToMV, or a distantly related potato virus X (PVX: genus Potexvirus) without induction of cell death (Bhattacharjee et al. 2009; Mestre and Baulcombe 2006; Sasaki et al. 2013, 2021). Using this transient expression assay system, we have demonstrated the essential role of N introns in the regulation of N-mediated resistance against TMV. Four introns in the N gene, particularly introns 1 and 2, are associated with transcriptional upregulation of N to effectively induce cell death in tobacco in the presence of p50 and N-mediated resistance in N. benthamiana after challenge with ToMV encoding GFP (Taku et al. 2018; Ikeda et al. 2021). Intron 3, which is required for full N-mediated resistance to TMV infection (Dinesh-Kumar and Baker 2000), produces an alternatively spliced variant encoding a C-terminally truncated N protein that can interfere with N function (Sasaki et al. 2013). The role of intron 4 remains to be elucidated.

In this study, we analyzed the RNA structure of intron 4 of the N gene and used computational analysis to identify a sequence within a myb domain protein 92 (MDP92)-encoding gene that could be targeted by a small RNA from the intron. While we did not detect an intron 4-derived small RNA with this sequence, we found the trend that the expression level of endogenous MDP92 was temporarily decreased in TMV-infected tobacco cultivars with and without the N gene. Studies of transient overexpression of MDP92 in N. benthamiana in the presence or absence of N, followed by challenge with ToMV or PVX, were conducted. Additionally, the effect of transiently overexpressed MDP92 on the expression levels of defense-related genes was determined.

Plants

Nicotiana tabacum cvs. Samsun NN (used as NN tobacco) and Samsun nn (used as nn tobacco) and N. benthamiana were used in this study. Seeds of tobacco and N. benthamiana were sown in Rockwool blocks (Nittobo, Tokyo, Japan) and Kumiai Nippi soil (Nihon Hiryo, Tokyo, Japan), respectively, and grown at 25°C under a 16 h light/8 h dark cycle for about 2 weeks. Seedlings were transferred to pots containing vermiculite (tobacco) and soil (N. benthamiana) and grown under the same conditions. Tobacco plants were fertilized once a week with 1,000-fold diluted Hyponex solution (Hyponex Japan, Osaka, Japan). Six- to eight-week-old plants were used for virus inoculation and agroinfiltration.

Target gene search

Using the BLAST search function of the Sol Genomics Network (SGN) (https://solgenomics.net/) and NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), we searched for mRNA sequences of N. tabacum, N. sylvestris, and N. tomentosiformis that were homologous to the 333-nt intron 4 sequence. We manually identified mRNA sequences that were complementary to one of the predicted small RNAs, including the MDP92 sequence (mRNA_127264 in the N. tabacum BX database of SGN).

Plasmids

pGLW3-erGFP and pGR107-erGFP contain the infectious clone of ToMV and PVX, respectively, each encoding ER-targeted GFP (erGFP) (Sasaki et al. 2013; Suzuki et al. 2024). pART27-35Sa-MDP92-HA and pART27-35Sa-GUS-HA for expression of MDP92 and GUS, respectively, were constructed as described in Supplementary materials and methods. Primers used for plasmid construction in this study are listed in Table S1. Expression of the GUS and MDP92 transgenes is driven by the 35S promoter.

Virus inoculation

Virions of the TMV OM strain were prepared and mechanically inoculated as described previously (Sasaki et al. 2012). NN and nn tobacco plants were inoculated with 1 µg and 100 ng of TMV virions in 10 mM sodium phosphate buffer (pH7.0), respectively, and maintained at 20°C for 3 days. Phosphate buffer alone was used for mock inoculation. In the temperature shift assay, inoculated NN tobacco plants were kept at 30°C for 40 h and then at 20°C for 8h.

Agrobacterium-mediated infiltration

Transformation, growth, and infiltration of Agrobacterium tumefaciens (Rhizobium radiobacter) strain GV3101(pMP90) were performed as described previously (Haque et al. 2009), except that transformed agrobacteria were grown in YEP medium (10 g/l Bacto pepton, 10 g/l Bacto yeast extract, and 5 g/l NaCl) and strain GV3101(pMP90) transformed with the pSoup helper plasmid (Hellens et al. 2000) was used for pGLW3-erGFP. Plants were incubated at 25°C after infiltration. The ratio of the final OD₆₀₀ value for the inoculum is summarized in Table S2.

Protoplast preparation and viral RNA inoculation

Protoplasts were prepared from N. benthamiana leaves at 48 h after infiltration of Agrobacterium transformants for MDP92 or GUS as previously described (Sasaki et al. 2009). pTocJ-GFP vector plasmid (Hori and Watanabe 2003) was linearized with MluⅠ and used for in vitro transcription with a HiScribe T7 in vitro transcription kit (New England Biolabs, Ipswich, USA). Virus inoculation was performed with 1 µg of transcribed RNA by the polyethylene glycol method (Kroner and Ahlquist, 1992). The inoculated protoplasts were incubated at 25 ℃ for 36 h in 0.6 M mannitol solution containing 1× Gamborg's B5 Medium Salt Mixture (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and 0.2 mg/ml chloramphenicol.

Fluorescence observation

An all-in-one fluorescence microscope BZ-X800 (Keyence, Osaka, Japan) was used for fluorescence observation of leaves infected with ToMV-erGFP and PVX-erGFP. The size of fluorescent infection sites was analyzed using ImageJ software (http://rsb.info.nih.gov/ij/).

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from the frozen leaves using TRI-Reagent (Cosmo Bio, Tokyo, Japan). Residual DNA in the total RNA was removed using recombinant DNaseI (Takara Bio, Kusatsu, Japan). cDNA was synthesized using PrimeScript RT regent Kit with gDNA Eraser (Perfect Real Time) (Takara Bio). TB Green Premix DimerEraser (Takara Bio) was used for RT-qPCR. Gene-specific primer sets used for qPCR are listed in Table S2. Expression levels of the MDP92 transgene were confirmed by RT-qPCR in N. benthamiana after agroinfiltration (Fig. S1).

Statistical analysis

Statistical analysis was performed on Microsoft Excel 2016 using the statistical software statcel3 (OMS Publishing, Saitama, Japan). The chi-square test was used to test the normality of the treatment values of all groups. For parametric statistics, Student’s t-test was performed. For data that did not show normality of variance, a non-parametric Wilcoxon signed-rank test was conducted.

Expression of the tobacco MDP92 gene tended to be suppressed in association with N gene activation

As a starting point for this study, we analyzed the secondary RNA structure of intron 4 of the N gene to estimate its possible role in N-mediated resistance, showing that its RNA sequence could contain stem-loop structures with a 21-bp perfectly complementary stem (Fig. S2a). It was possible that this predicted stem could produce small RNAs to regulate defense responses. As a result of database searches for the potential targets of possible intron 4-derived small RNAs, we found that a tobacco MYB-related mRNA (mRNA_127264) contained the 21-nt sequence in the 3’-UTR, which was complementary to the intron 4 sequence (Fig. S2b). The mRNA encodes a protein annotated as MDP92, which contains two helix-loop-helix (HLH) domains conserved in the R2R3-type MYB transcription factor (Fig. S3a and b). We also found other potential targets within non-functional genomic regions, such as an extremely long 5'-UTR, within an intron, or in a pseudogene (data not shown), which we decided not to analyze.

On the assumption that the possible intron 4-derived small RNAs could affect MDP92 expression, RT-qPCR with a temperature shift assay was performed to investigate whether MDP92 expression changes during N activation upon TMV infection. HR was synchronously induced by shifting to 20 ˚C after 40 h of incubation of TMV-inoculated NN tobacco at 30 ˚C, as the N gene is inactivated at 30 ˚C and activated at 20 ˚C (Wright et al. 2000). In TMV-inoculated plants, transcript levels of N increased sharply from 2 h after temperature shift (hat), peaked at 4 hat, and gradually decreased thereafter, while they were relatively constant in mock-inoculated plants (Fig. 1a and b). On the other hand, in both TMV- and mock-inoculated plants, transcript levels of MDP92 decreased from 0 to 6 hat (Fig. 1c). Although we did not find a statistically significant difference, the MDP92 transcript level in TMV-inoculated plants was higher at 0 (1.1-fold) and 2 (1.2-fold) hat, but lower at 4 (0.73-fold), 6 (0.70-fold), and 8 (0.87-fold) hat than that in mock-inoculated plants (Fig. 1d). These results led us to speculate that MDP92 expression might be downregulated in response to temperature change and further suppressed in association with N activation.

To eliminate the effect of temperature change on MDP92 expression, we then examined NN tobacco plants maintained at the constant temperature of 20 ˚C after inoculation with TMV. N transcript levels in TMV-inoculated plants continued to increase from 0 to 3 days after inoculation (dai) as virus infection progressed (Fig. 1e, f, and i). MDP92 transcript levels were similar between TMV- and mock-inoculated plants at 0, 2, and 3 dai (Fig. 1g and h). However, there was a different expression pattern at 1 dai, where MDP92 transcript levels increased in mock-inoculated plants but remained unchanged in TMV-inoculated plants (Fig. 1g and h). Based on these results, we hypothesized that MDP92 expression without TMV infection might temporarily be upregulated in response to wounding by inoculation but suppressed during the early stage of N activation induced by TMV infection.

We conducted RNA-Seq analysis of small RNAs in TMV-infected NN tobacco plants and detected known N-related miRNAs and small RNAs (Li et al. 2012), but not the intron 4-derived small RNAs (Table S3). Therefore, we did not show that small RNAs were produced from intron 4 of the N gene under the current experimental conditions.

Transient overexpression of MDP92 promoted ToMV intercellular movement in N. benthamiana leaves that induced N resistance

Our finding that MDP92 expression tended to be suppressed during N activation raised the possibility that MDP92 might have an impact on N-mediated virus resistance. To investigate this possibility, we performed a transient expression assay by agroinfiltration in N. benthamiana. In this assay, an MDP92 coding sequence was transiently expressed with an infectious cDNA of a ToMV variant encoding ER-targeting GFP (ToMV-erGFP) together with the N coding sequence including its introns (gN-Int1234). A GUS cDNA was used as a negative control. N. benthamiana was used to evaluate the effect of N-mediated virus resistance without inducing visible cell death (Bhattacharjee et al. 2009; Mestre and Baulcombe 2006; Sasaki et al. 2013; 2021).

At 72 h after infiltration (hai), the size of individual fluorescent infection sites and virus accumulation per leaf were significantly higher in MDP92-expressing leaves than GUS-expressing leaves (Fig. 2a, b, and d). Meanwhile, the average number of fluorescent infection sites was similar between the two treatments (Fig. 2c). These results suggested that transient overexpression of MDP92 enhanced virus intercellular movement during N-mediated resistance.

Different expression patterns of the endogenous MDP92 gene between TMV-infected and uninfected nn tobacco plants

Since nn tobacco also has the MDP92 gene, it was possible that MDP92 might be involved in N-independent basal resistance. To test this possibility, we compared MDP92 expression patterns between nn tobacco plants with and without TMV infection. MDP92 transcript levels in mock-inoculated plants were slightly higher at 1, 2, and 3 dai than at 0 dai, possibly due to the response to wounding (Fig. 3a). Compared to mock-inoculated plants, MDP92 transcript levels in TMV-inoculated plants were significantly lower at 1 dai, but similar at 2 and 3 dai (Fig. 3a). The transcript levels of MDP92 in TMV-inoculated plants at 1, 2, and 3 days after inoculation were 0.6, 1.4, and 1.3 times those in the control plants, respectively (Fig. 3b). Virus RNA levels increased sharply between 2 and 3 dai (Fig. 3c). These results suggest that, similar to the results for NN tobacco, the expression of MDP92 in nn tobacco, which would be upregulated by wounding in the absence of TMV infection, may need to be suppressed during the early induction of basal resistance to the virus, but later regulated to levels comparable to those in control plants.

Transient expression of MDP92 increased ToMV infection sites in N. benthamiana

To further elucidate the role of MDP92 in virus resistance in the absence of the N gene, we examined the effect of transient expression of MDP92 or GUS on ToMV-erGFP infection in N. benthamiana. The number of fluorescent infection sites in MDP92-expressing leaves was significantly higher than that in GUS-expressing leaves at 48 hai, but not at 72 hai (Fig. 4a and c). Consistently, the virus accumulation level in MDP92-expressing leaves was significantly higher than that in GUS-expressing leaves at 48 hai (Fig. 4d). However, the fluorescence size of individual infection sites was similar between the two treatments (Fig. 4b). These results suggested that transient overexpression of MDP92 enhanced virus amplification in initially infected cells. We then performed an inoculation assay using protoplasts transiently overexpressing MDP92 or GUS. For inoculation, we used in vitro transcripts of TocJ-GFP, which is a chimeric ToMV encoding GFP and the CP of tobacco mild green mosaic virus (Hori and Watanabe 2003). RT-qPCR analysis showed that viral RNA levels were twice as high in MDP92-expressing protoplasts as in GUS-expressing protoplasts (Fig. 4e). These collective data suggested that overexpressed MDP92 may attenuate basal resistance that limits virus amplification at the single cell level.

Different effects of transient overexpression of MDP92 in the presence or absence of N resistance on the expression of defense-related genes

Based on the finding that transient overexpression of MDP92 can negatively affect N resistance and basal resistance in N. benthamiana, we investigated its effect on the expression of selected defense-related genes (i.e. NbPR1a, NbPR4, NbHin1, and NbHsr203j). NbPR1a and NbPR4 were selected as marker genes for the salicylic acid (SA) and jasmonic acid (JA) signaling pathways, respectively (Diao et al. 2019; Pascual et al. 2015). NbHin1 and NbHsr203j were used as resistance-related markers (Peng et al. 2019; Takagi et al. 2022). RT-qPCR analysis was performed on ToMV-erGFP-infected leaves expressing MDP92 or GUS with or without N resistance induction. Regardless of N resistance, transient overexpression of MDP92 did not affect the transcript level of NbPR1a at 48 or 72 hai (Fig. 5a and e). For NbPR4 (Fig. 5b and f), NbHin1 (Fig. 5c and g), and NbHsr203j (Fig. 5d and h), there were some cases where the transcript levels were significantly different between MDP92 and GUS-expressing leaves. In the presence of N resistance, the transcript level of NbHsr203j was significantly lower in MDP92-expressing leaves than in GUS-expressing leaves at 48 hai (Fig. 5d). In the absence of N resistance, the transcript levels of both NbPR4 and NbHin1 were significantly higher at 48 hai in MDP92-expressing leaves than in GUS-expressing leaves (Fig. 5f and g). These results suggested that MDP92 is differently involved in the regulation of these defense-related genes during N resistance and basal resistance.

To confirm that MDP92 functions as a nuclear transcription factor, we examined the subcellular localization of YFP-fused MDP92 (MDP92-YFP). At 24 hai, MDP92-YFP was localized only in the DAPI-stained nucleus (Fig. S4) in N. benthamiana. This suggested that MDP92 may be a transcription factor that regulates gene expression in the nucleus.

Transient overexpression of MDP92 enhanced PVX infection sites in N. benthamiana

To further investigate whether MDP92 affects basal resistance to viruses other than ToMV, PXV-erGFP was inoculated to N. benthamiana leaves transiently expressing MDP92 or GUS. The average number of fluorescent sites and the level of virus accumulation in MDP92-expressing leaves were significantly higher than in GUS-expressing leaves at 48 hai (Fig. 6a, c, and d). The average size of individual fluorescent sites was comparable between MDP92- and GUS-expressing leaves at 48 and 72 hai (Fig. 6b). In addition, gene expression analysis of defense-related genes during PVX-erGFP infection showed that the significant difference in transcript levels was observed for NbPR4 and NbHin1 at 48 hai (Fig. 6f and g), but not for NbPR1a and NbHsr203j (Fig. 6e and h). These results suggested that MDP92 plays a role in attenuating basal resistance to the early infection with PVX as well as ToMV, which is associated with NbPR4 and NbHin1 expression.

In addition, we performed RT-qPCR to examine the expression levels of an N. benthamiana orthologous gene to MDP92, NbMDP92 (Fig. S3c, Table. S4), using total RNA from the samples with or without the transient expression of MDP92 and/or infection with ToMV-erGFP or PVX-erGFP. However, little or no expression of endogenous NbMDP92 was detected in any of the samples tested (data not shown). These results suggested that NbMDP92 expression is always close to the detection limit and does not affect our results from the MDP92 transient expression experiments described above.

MYB transcription factors have been shown to play important roles in the regulation of various physiological and biochemical processes in plants, including defense responses to pathogens (Du et al. 2009; Dubos et al. 2010), but limited information is available on MYB function in virus resistance (Biswas et al. 2023; Viswanath et al. 2023). In this study, we found the trend that the expression levels of the endogenous MDP92 gene, which encodes an R2R3-type MYB transcription factor, were temporarily lower in both NN and nn tobacco plants infected with TMV compared with mock-treated plants, raising the possibility that MDP92 may negatively regulate virus infection. In support of this idea, transient overexpression of MDP92 in N. benthamiana enhanced intercellular movement of ToMV in the presence of N resistance induction and increased the number of infection sites and virus accumulation per leaf during early infection with ToMV or PVX in the absence of N resistance induction. Our protoplast inoculation assay further showed that transient overexpression of MDP92 increased virus accumulation at the single cell level. MDP92 localization to the nucleus was also confirmed. These results suggest a possibility that MDP92 may play an attenuating role in N resistance and basal resistance to viruses as a transcription factor.

The interplay between the SA and JA signaling pathways is antagonistically or cooperatively associated with the induction of resistance to tobamovirus infection in Nicotiana species, depending on the experiment (Kobayashi et al. 2010; Oka et al. 2013; Shang et al. 2011; Zhu et al. 2014). In this study, we examined whether either or both of the SA and JA signaling pathways are positively or negatively affected by overexpressed MDP92. Unexpectedly, in the presence of N, we did not find significant differences in the transcript levels of NbPR1a, NbPR4, or NbHin1 between MDP92- and GUS-expressing tissues, except for NbHsr203j at 48 hpi with ToMV-erGFP. This result suggests that neither the SA nor the JA signaling pathway is essential for the observed increased virus intercellular movement, while an unidentified defense response represented by NbHsr203j expression is negatively affected by MDP92 overexpression. In our previous study of N resistance induced by co-expression of N and p50 in N. benthamiana, NbPR1a transcript levels were similar between co-expression and control treatments (Sasaki et al. 2021). The same study also showed that N resistance, which caused strong inhibition of the intercellular movement of ToMV-erGFP, was associated with stimulation of ROS production, an indicator of defense response induction (Sasaki et al. 2021). Since phytohormones such as brassinosteroids and abscisic acid, in addition to SA and JA, are involved in ROS production (Ali et al. 2023; Xu et al. 2024), it will be interesting to investigate whether gene regulation related to ROS production and signaling is involved in N-mediated responses specific to virus intercellular movement in N. benthamiana and is also affected by MDP92 overexpression independently of the SA and JA signaling pathways.

On the other hand, with respect to N-independent basal resistance to ToMV-erGFP and PVX-erGFP, the expression of NbPR4 and NbHin1 was significantly upregulated in MDP92-expressing leaves at 48 hpi, whereas the expression of NbPR1a and NbHsr203j was not affected. Thus, MDP92 is involved in activating a cascade of the JA signaling pathway without affecting the SA signaling pathway, possibly leading to attenuate basal resistance to virus amplification at the early infection stage. A JA-mediated relationship between NbHin1 and basal resistance is shown by the recent finding that overexpression of NbHin1in N. benthamiana induces expression of the Rab11 gene, which is involved in JA biosynthesis, and enhances TMV resistance at the late infection stage (i.e. local and systemic spread) (Peng et al. 2019), whereas it is unclear whether NbHin1 overexpression affects virus amplification during early infection. To better understand the attenuating effect of MDP92 function on basal resistance, it is important to comprehensively investigate how the expression profiles of genes related to defense responses, particularly the JA-signaling pathway, are altered when MDP92 is overexpressed.

From our collective results in this study, we hypothesize that the MDP92 transcription factor may be transcriptionally downregulated at the early stage of resistance induction to compromise its negative impact on virus resistance. In contrast to MDP92, the expression of NtMYB1 in NN tobacco and NbMYB1 and NbMYB4L in N. benthamiana was reported to be upregulated upon TMV infection, and gene silencing of NbMYB1 in transgenic N. benthamiana carrying the N gene and that of NbMYB4L in wild-type N. benthamiana were shown to attenuate N-dependent resistance and basal resistance, respectively (Liu et al. 2004; Yang and Klessig, 1996; Zhu et al. 2022). In addition, the expression of NtMYB2, an NtMYB1 homolog, was induced in NN tobacco by chitin treatment and wounding, suggesting its contribution to responses to biotic and abiotic stresses (Sugimoto et al. 2000). The amino acid alignment between MDP92 and NtMYB1 and its homologs (NtMYB2, NtMYB3, and NtMYB4) as well as NbMYB1 and NbMYB4L shows that two HLH domains in the N-terminal region are highly conserved (Fig. S3b), whereas phylogenetic analysis of MYB proteins shows that MDP92 is in a separate clade from NtMYB1-4, NbMYB1, NbMYB4L, and NbMDP92 (Fig. S3c). These results suggest that the function and gene regulation of MDP92 during virus infection are distinct from these other MYB proteins.

This study also shows that in mock-inoculation, the transcript level of MDP92 continued to decrease until 6 h after the temperature shift or increased every other day when inoculated plants were grown at constant temperature. Therefore, MDP92 may play other roles in responses to abiotic stresses, including temperature change and wounding. AtMYB92 of Arabidopsis thaliana in the same clade as MDP92 (Fig. S3c) activates the expression of genes related to fatty acid biosynthesis, when constitutively overexpressed in N. benthamiana (To et al. 2020). Interestingly, fatty acids are involved in plant defense responses and virus replication (Fang et al. 2024; Walley et al. 2013). Further studies of MDP92 are needed to determine its target genes related to biotic and abiotic stress responses and fatty acid biosynthesis and metabolism, which may provide a clue to explain how MDP92 is involved in the regulation of gene expression that affect viral intercellular movement and replication processes.

Acknowledgments

We thank Dr. Richard S Nelson and Dr. Ken Komatsu for valuable comments on this manuscript. This work was supported by the FLOuRISH Fellowship Program for Next Generation Researchers at the Tokyo University of Agriculture and Technology (to M.Y.) and partly by the Japan Society for Promotion of Science (JSPS) KAKENHI Grant Numbers 26450052 and 19K06047 (to N.S.). We also thank the Institute of Global Innovation Research at Tokyo University of Agriculture and Technology (GIR-TUAT) for the Special Research Fund.

Author contributions

M.Y. and N.S. conceived and designed the study. M.Y., R.S., and T.S. prepared materials and performed the experiments. M.Y., R.S., Y.M., and N.S. analyzed the data. M.Y. and N.S. wrote the manuscript with edits and additional text from Y.M.

Conflict of interest

The authors declare no conflicts of interest.

Human and animal rights

This article does not contain any studies involving human participants or experimental animals.

Compliance with Ethical Standards

This article does not contain any studies with human participants or animals performed by any of the authors.

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Enhanced virus infection in Nicotiana benthamiana transiently overexpressing MDP92 , encoding a tobacco MYB transcription factor

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Abstract

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Introduction

Materials and methods

Plants

Target gene search

Plasmids

Virus inoculation

Agrobacterium-mediated infiltration

Protoplast preparation and viral RNA inoculation

Fluorescence observation

Reverse transcription-quantitative PCR (RT-qPCR)

Statistical analysis

Results

Discussion

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References

Supplementary Files

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