Throughout their life cycle, plants are continuously challenged by various types of pathogens. To cope with these challenges, plants evolved a two-tiered immune system that enables them to mitigate the effects of invading pathogens (Jones and Dangl, 2006; Delplace et al., 2020). Following pathogen infection, plants can develop induced resistance to cope with future attacks. Induced resistance is generally divided into systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Walters and Heil, 2007). SAR, commonly triggered by local infection, can provide long-term resistance to subsequent infection (Klessig et al., 2018). SAR generally relies on salicylic acid (SA), which has been shown to increase in plant tissues during pathogenesis (Yalpani et al., 1991).

Countless studies in the past few decades have focused on elucidating the interactions between various plant hosts and the pathogens that threaten them, however, most of these studies investigate the interaction between the plant host and a single pathogen, in controlled conditions. In agricultural or natural environments, plants are routinely exposed to more than one pathogen at a given time. Such infections can be challenging to mimic in research settings. Some studies have attempted to elucidate the nature of particular diseases caused by a combination of pathogens (Lamichhane and Venturi, 2015; Fang et al., 2021; Tan et al., 2021). In some cases, one pathogen can prime immunity against another (Orton and Brown, 2016), while in other cases, a prior or simultaneous pathogen infection will increase plant susceptibility towards additional pathogens (Spoel et al., 2007; Cooper et al., 2008; Zhang et al., 2022b).

Plant viruses of the genus Tobamovirus (family Virgaviridae) are some of the most economically important plant pathogens. Tobamoviruses are highly infectious, and cause significant damage to various crop species. They are transmitted by mechanical contact: through e.g., workers’ hands, agricultural tools and soil (Broadbent, 1976), and the diseases symptoms caused by these viruses include stunting, leaf blistering, malformations and yellow mosaic patterns, fruit spotting and marbling and suppression of root branching (Luria et al., 2017; Vaisman et al., 2022). The tomato mosaic virus (ToMV) was isolated in the early 20^th century as a virus that severely impacts tomato crops (Broadbent, 1976). Since then, effective ToMV-resistance genes have been discovered in tomato, and currently most modern cultivars are immune to this virus (Hall, 1980). In recent years, the tobamovirus tomato brown rugose fruit virus (ToBRFV) (Zhang et al., 2022a) has caused significant damage to tomato crops, leading to reductions in fruit quality and yield in various parts of the world. This is largely due to the ability of the ToBRFV movement protein to overcome the durable tomato resistance gene, Tm-2² (Luria et al., 2017; Hak and Spiegelman, 2021). Since there is currently no available commercial genetic resistance to ToBRFV, the only means to control ToBRFV is by effective detection, containment and disinfection (Oladokun et al., 2019; Panno et al., 2019; Davino et al., 2020; Alon et al., 2021; Samarah et al., 2021; Dombrovsky et al., 2022).

In many cases, viral infection has been reported to exacerbate the symptoms of another pathogen infection, leading to more severe disease (Bains and Parkash, 1984; Omar et al., 1986; Meyer and Pataky, 2010; Tollenaere et al., 2017; Philosoph et al., 2018). For example, the tobamovirus cucumber green mottle mosaic virus (CGMMV) has been shown to increase the severity of Pythium wilting in cucumber under various environmental conditions (Philosoph et al., 2018; Philosoph et al., 2019). Other outcomes of co-infection with viruses and phytopathogens have been reported, with viral infection reducing disease susceptibility in certain cases (Latch and Potter, 1977; Varughese and Griffiths, 1983). However the molecular pathways determining these interactions remain unclear.

Botrytis cinerea (B. cinerea, Bc) is the causative agent of gray mold disease (Williamson et al., 2007). B. cinerea infects upwards of 1400 plant hosts, many of them crop plants, and causes severe damage in greenhouses and post-harvest storage and transport (Williamson et al., 2007; Elad et al., 2016; Fillinger and Elad, 2016). Xanthomonas euvesicatora (Xcv) causes bacterial leaf spot (BLS) on peppers and tomatoes. BLS severely reduces plant productivity and yield (Jones et al., 2004). Since B. cinerea and X. euvesicatoria are important pathogens in tomato agricultural cropping (Moss et al., 2007; Fillinger and Elad, 2016), and since the tobamovirus ToBRFV has become widespread in worldwide tomato agriculture (Zhang et al., 2022a), we were interested to examine the impact of the interaction between B. cinerea/X. euvesicatoria and tobamoviruses on tomato disease response.

Different pathogens can activate common or different pathways in their plant host. The nature of the defense pathways activated during infection with more than one pathogen will likely influence the eventual resistance or susceptibility to each pathogen. B. cinerea, primarily a necrotrophic plant pathogen, is known to activate Jasmonic acid (JA) pathway defenses (Rowe et al., 2010; Mehari et al., 2015; Fillinger and Elad, 2016). Mutant plants with impaired JA content or sensing are notoriously sensitive to B. cinerea infection (AbuQamar et al., 2008; Gupta et al., 2021a; Gupta et al., 2022). B. cinerea was also demonstrated to activate SA-dependent pathways in planta, promoting pathogenesis (El Oirdi et al., 2011; Rossi et al., 2011). Tobamoviruses are known to activate a different plant defense response pathway, the salicylic acid (SA) pathway (White, 1979; Gaffney et al., 1993; Murphy and Carr, 2002). While JA confers defense against necrotrophic pathogens, SA is largely thought to confer protection against biotrophic pathogens (Yang et al., 2015). The competitive crosstalk between the JA and SA pathways determines the precise defense response to the specific attacker (Thaler et al., 2012; Caarls et al., 2015; Aerts et al., 2021). In this work, we found that infection with the tobamoviruses ToMV or ToBRFV resulted in increased B. cinerea incited disease, likely through increases in endogenous SA and activation of the SA pathway. Our results suggest that manipulation of plant defense pathways by viruses may lead to unexpected outcomes, which include increased susceptibility to additional pathogens. Given that the increased pathogen susceptibility may ultimately cause more damage than the original virus, future management strategies for plant viruses should take into account both the primary damage caused by viruses, and the secondary effect of plant viruses on susceptibility to additional pathogens such as B. cinerea.

Plants often encounter multiple pathogens in their environment (Abdullah et al., 2017). In addition to possible interactions between two or more pathogenic microorganisms on or within the plant, the interaction of each pathogen with the plant host can shape the hosts’ subsequent response to additional pathogens. Interactions between plant-infecting microorganisms can be symbiotic, synergistic, or antagonistic, either directly, or through the plant host (Abdullah et al., 2017). In our work, we found that the tobamoviruses ToMV and ToBRFV cause aggravation of the disease caused by the necrotrophic fungal pathogen B. cinerea, and moderation of the disease caused by the hemibiotrophic bacterial pathogen X. euvesicatoria ( Figure 2 ). These interactions are likely generated by the response of the tomato host, through increases in SA levels ( Figure 5 ), which aggravated the symptoms caused by the necrotroph B. cinera and attenuated the symptoms of the biotroph X. euvesicatoria. Comparison between the two studied tobamoviruses demonstrates that ToMV promoted greater increases in SA levels, stronger susceptibility to B. cinerea, and resistance to X. euvesicatoria, while ToBRFV promoted milder increases in SA levels, weaker susceptibility to B. cinerea, and no resistance to X. euvesicatoria ( Figures 2 , 5 ). Exogenous SA treatment also resulted in susceptibility to B. cinerea elicited disease and resistance to X. euvesicatoria elicited disease ( Figure 5 ), suggesting that these differential responses observed in virus-infected plants are due to increased SA levels. In addition, NahG plants lacking endogenous SA were not able to respond to tobamovirus infection with increases in B. cinerea susceptibility or X. euvesicatoria resistance ( Figure 6 ). Taken together, these findings support the hypothesis that the effects of viral infection on pathogen susceptibility observed in our system indeed involve SA-regulated pathways. It is important to note that when studying dual infections, the relative timing of both infections may alter the experimental outcome. In our system, the second pathogen infection occurred after systemic viral infection had occurred (21 dpi) and the virus was fully established in its host. It is possible that co-infection at earlier stages, for example during the first days of infection when the virus had not yet moved out of the infected leaf, may yield different results.

SA promotes resistance to many plant pathogens, including bacteria, fungi and viruses (Peng et al., 2021), and is required for plant immunity to (hemi)biotrophic pathogens. SA-mediated immunity has typically been described as occurring via activation of the SAR pathway (Fu and Dong, 2013; Di et al., 2017). Interestingly, early investigations of plant response to the tobamovirus TMV uncovered that SA promotes plant resistance to TMV (White, 1979). Plants which are unable to accumulate SA are highly susceptible to TMV (Gaffney et al., 1993), as SA reduces viral accumulation and cell-to-cell movement (Murphy and Carr, 2002). Tobamovirus infected plants were previously shown to accumulate high levels of SA (Malamy et al., 1990; Conti et al., 2017). We also observed hyper-accumulation of endogenous SA contents in ToMV- and ToBRFV- infected plants ( Figure 5 ). Previously, we demonstrated that the ability of ToBRFV to break Tm-2² resistance is linked to its moderated mobility (Hak and Spiegelman, 2021; Hak et al., 2023), suggesting that ToMV spreads faster than ToBRFV in a tobamovirus-susceptible background. However, since there is currently no genetic resistance to ToBRFV in tomato, it poses a much greater agricultural challenge. Interestingly, our current work demonstrates that the reduced movement of ToBRFV (Hak and Spiegelman, 2021) correlated with a lower ability to promote increases in plant SA, as compared with ToMV ( Figure 5 ). Thus, it would seem that systemic plant movement is correlated with the plants’ SA production, and the plant activates its SA pathways at a level commensurate with the infectivity of the virus attacking it.

Elevated SA levels are known to promote susceptibility to necrotrophic pathogens, including B. cinerea (Mengiste, 2012). Exogenous SA treatment, and endogenous SA accumulation following biotrophic pathogen infection, were both previously demonstrated to increase susceptibility to the necrotrophic fungal pathogen A. brassicicola in Arabidopsis (Spoel et al., 2007), due to inhibition of JA dependent defenses by SA. Loss of Arabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) enhanced B. cinerea susceptibility in an SA-dependent manner (Veronese et al., 2006), and B. cinerea is known to activate the SA pathway in host plants (El Oirdi et al., 2011; Rossi et al., 2011). Suppression of the tobacco mitogen-activated protein kinase phosphatase (NtMKP1) negatively regulated SA-mediated tissue necrosis and as a result enhanced resistance to B. cinerea (Oka et al., 2013). Furthermore, viral infection has been demonstrated to promote susceptibility to several necrotrophic pathogens, including B. cinerea (Omar et al., 1986; Meyer and Pataky, 2010; Philosoph et al., 2018). Our results suggest that the tobamovirus-mediated hyper-accumulation of SA may increase susceptibility to B. cinerea elicited disease in a similar manner.

While SA is considered to promote resistance against viruses, in some cases, SA fails to suppress viral infection (Murphy et al., 2020). In fact, several viruses promote SA biosynthesis and signaling during systemic infection (Krečič-Stres et al., 2005; Zhou et al., 2014; Shaw et al., 2019). It is therefore possible that while SA accumulates during viral infection, viruses suppress specific elements downstream to SA to successfully infect the plant. For example, the tobamovirus movement protein suppresses callose deposition near plasmodesmata (Guenoune-Gelbart et al., 2008), an SA- dependent process (Wang et al., 2013). The coat protein suppresses the expression of several SA-responsive genes to allow systemic transport of the virus (Conti et al., 2012; Venturuzzi et al., 2021). Other SA-mediated responses, which are not suppressed by the virus, would likely remain activated (Laird et al., 2013; Yang et al., 2016). Alternatively, the upregulation of SA could reflect a failed defense response, which is initiated after the virus is already established. This type of dysfunctional immune response may cause deleterious effects, including enhanced disease symptoms. Indeed, several studies have shown that strong viral symptoms are often associated with upregulation of the SA-responsive genes and metabolites (Diaz-Vivancos et al., 2006; Hernández et al., 2006; Niehl et al., 2006; Kogovšek et al., 2010; Shang et al., 2010). These observations suggest that during viral infection, the upregulation of several SA-mediated pathway elements may enhance the disease symptoms rather than protect plants against the virus. We propose that another effect of this immune activation may be the alteration of plant response to other pathogens.

Reportedly, SAR depends on the SA pathway, while ISR relies on JA/ET pathways. While SA is antagonistic to JA in defense signaling (Pieterse et al., 2009; Pieterse et al., 2012), there are cases where SA and JA were reported to positively interact (Ferrari et al., 2003; De Vleesschauwer et al., 2013; Liu et al., 2016; Ullah et al., 2022). Dependence of ISR on SA signaling has been reported as well (Martínez-Medina et al., 2013). Distinctions between ISR and SAR are not clear cut in tomato, and overlap between the two was reported (Liu et al., 2016; Betsuyaku et al., 2018). Thus, while plant defense against B. cinerea requires the JA-mediated pathway (AbuQamar et al., 2008), and plant defense against viruses involves the SA-mediated pathway (White, 1979; Malamy et al., 1990), we found that the plants response to tobamoviruses and B. cinerea operates in the same direction, with virus-infected plants becoming more susceptible to B. cinerea, in a SA-dependent manner ( Figures 2 ; 5 - 6 ). This is likely due to antagonism between the high levels of SA elicited by the viral infection ( Figure 5 ), and JA required to promote plant defenses against B. cinerea.

It should be noted that the SA-mediated effects on B. cinerea pathogenesis may also depend on the specific concentration of SA. For example, low levels of the SA analog acibenzolar-S-methyl promoted resistance to B. cinerea in tomato (Leibman-Markus et al., 2023), and priming SA-mediated defenses was also found to induce B. cinerea resistance (Audenaert et al., 2002; Gupta et al., 2020; Gupta et al., 2022; Marash et al., 2022). Here, ToMV infection resulted in SA levels of approximately 1000 ng/gr, which is equivalent to ~8mM. However, exogenous application of 5 mM resulted in phytotoxicity. Interestingly, we found that during tobamovirus infection, the levels of the SA receptor NPR1 are down-regulated ( Figure 4A ). This negative feedback mechanism may curb the SA response even when SA levels are exceedingly high, and may explain how the plant tolerates high endogenous doses of SA during infection. Nevertheless, the excess levels of SA may still create conditions that are favorable for necrotrophs by promoting cell death.

Our results lead to the conclusion that in certain cases, co-infection with biotrophs and necrotrophs, or SA-dependent and JA-dependent pathogens, could be particularly devastating, because the strong plant response to one type of pathogen makes the host unable to properly respond to another type of pathogen. This could have implications both in co-infection research and in agricultural cultivation. Different pathogens can be inexplicably aggressive in particular growing seasons, and it is plausible that one of the underlying causes of this might be strong activation of one pathway of the plant immune or stress response system, leaving the plant unable to respond to subsequent challenge. However, this is only relevant to a sub-set of phytopathogens and plant host systems, as viral infection was also reported to increase susceptibility to biotrophic pathogens (Latch and Potter, 1977; Bains and Parkash, 1984), and SA and JA are known to have both positive and negative interactions in plant defense (Takahashi et al., 2004; Li et al., 2019; Vlot et al., 2021; Ullah et al., 2022). These findings need to be taken into consideration when designing induced resistance protocols to mitigate plant viruses. While SAR-inducing compounds, such as acibenzolar-s-methyl were found to be effective against some viruses (Palukaitis et al., 2017), they may have a negative effect on the disease response to other pathogens, when co-infection occurs. The use of specific resistance inducers should therefore be tailored according to the needs of the specific crop, taking into account the additional potential pathogens within each specific cropping environment.

Based on our results it seems that tobamoviruses, which pose a substantial economic problem in worldwide agriculture (Zhang et al., 2022a), can also increase crop losses by making plants more susceptible to other classes of pathogens. This could be possibly mitigated by the fact that the same virus or viruses can generate some resistance to other classes of pathogens. Thus, the balance between pathogen resistance and pathogen susceptibility in a particular growing season, when dealing with tobamovirus infected plants, will depend on the prevalence of particular pathogens in the cropping environment. Our results indicate that resistance or susceptibility to different classes of pathogens in tobamovirus-infected plants are primarily a result of substantial changes to the SA pathway. Further research is needed to elucidate the relationships between different defense pathways during tobamovirus infection, as well as the roles of SA in B. cinerea pathogenesis and its interactions with the plant host.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

ZS and MB: conceptualization. ML-M, RG, DW, ZS and MB: methodology. ML-M, RG, and DW: investigation. ML-M, RG, DW, ZS and MB: analysis. ZS and MB: writing – original draft. ML-M, RG, DW, ZS and MB: writing – review and editing. ZS and MB: funding acquisition. All authors contributed to the article and approved the submitted version.