Plant defense against virus diseases; growth hormones in highlights

ABSTRACT

Phytohormones are critical in various aspects of plant biology such as growth regulations and defense strategies against pathogens. Plant–virus interactions retard plant growth through rapid alterations in phytohormones and their signaling pathways. Recent research findings show evidence of how viruses impact upon modulation of various phytohormones affecting plant growth regulations. The opinion is getting stronger that virus-mediated phytohormone disruption and alteration weaken plant defense strategies through enhanced replication and systemic spread of viral particles. These hormones regulate plant–virus interactions in various ways that may involve antagonism and cross talk to modulate small RNA (sRNA) systems. The article aims to highlight the recent research findings elaborating the impact of viruses upon manipulation of phytohormones and virus biology.

1. Introduction

Plant viruses utilize numerous strategies that are more conducive for replication and viral spread inside the plant’s cellular environment.^1–5 Phytohormone accumulation and signaling pathways can be directly or indirectly disrupted due to virus infections.⁶ Several phytohormone pathways exist within plants, contributing to plant growth, reproduction, and development.^7,8 Ethylene (Et), jasmonic acid (JA), and salicylic acid (SA) play an important role in mechanisms of defense.^9–11 However, abscisic acid (ABA), auxin (Aux), cytokinins (CK), gibberellins (GA),^12–16 and brassinosteroids (BR) also show defense relations but play vital roles in plant physiology and development.^17–19 Additionally, there is ‘cross talk’ and extensive interactions between different pathways of phytohormones,^20,21 providing information to regulate environmental cue responses or attack of pathogens.^22–24 Here, we discuss the role of various phytohormones in disease symptom development, host defenses, and enhanced virus replication and movement. We specifically focus upon plant–virus interactions associated with hormonal production, manipulations, and signaling.

2. Mechanisms for symptom development

Plant virus attack exhibits some common symptoms including stunting, curling of leaves, mosaic, mottle, and chlorosis.^25,26 Moreover, plant viruses have a great impact on the production of plant hormones leading towards the development of symptoms.²² Despite our understandings about the associations of viral components affecting the phytohormone pathways, our knowledge about their role in the development of symptoms is quite limited.²⁷ Recently, several plant–viral components were found involved in phytohormonal pathways linked to symptom development^6,28 (Figure 1). The first mechanistic explanation regarding modulation of virus regulatory systems through phytohormones within their host plants was provided by Wang et al. and Nafisi et al.,^46,47 which linked the symptom development to plant–virus interactions. Aux is an important phytohormone that is disrupted directly by viral components.⁴⁸ Aux signaling disruption largely depends upon the biosynthesis of Aux or mutants showing resembling symptoms to viral disease, i.e., stunting, leaf curling, mosaic, and mottle.^49,50 For example, Aux signaling has been shown to disrupt the Tobacco mosaic virus (TMV) 126-kDa replication protein via Aux/indole acetic acid (IAA) family member’s interaction.^51,52 Aux/IAA proteins act as negative regulators of Aux-responsive transcription factors (ARFs) to control and modulate genetic ability that is important in different plant functions.^53–55 TMV 126 kDa protein interaction disrupts Aux/IAA nuclear-localized proteins and results in the development of diseases such as leaf curling in different plants.^29,56 In contrast, TMV-V1087I or a related Tobacco mild green mosaic virus mutant shows no interaction with Aux/IAA proteins and results in symptoms of attenuated disease even though wild-type (WT) plants show replication of these viruses with considerable TMV levels.^29,52 Thus, the interaction between the TMV–Aux/IAA is strongly correlated with disease development process.^57,58 The viral silencing suppressor’s activity has also been linked to the Aux signaling and disease symptom development.⁵⁹ Specifically, transgenic Arabidopsis plants constitutively expressing turnip mosaic virus (TuMV) silencing via suppressor HC-Pro exhibit abnormal leaf development resembling the symptoms of viral diseases.³⁰ HC-Pro overexpression was found to increase several miRNA accumulations which include the targeting ARFs. Furthermore, these increased miRNA levels correspond to enhanced target cleavage.^30,60–64 Researchers claim that the expression of viral suppressing proteins interfering Aux regulatory pathways accounts for the development of many symptoms induced during viral infections.⁶⁵ GA biosynthesis pathway disruption is also closely related to symptoms of viral infections. GA takes part in cell division and elongation.⁶⁶ Stunting and leaf darkening symptoms in GA-deficient rice mutants are characteristically induced by Rice dwarf virus (RDV).⁶⁷ Specific interaction between the rice ent-kaurene oxidase (EK-O) and the RDV-P2 (an outer capsid protein) has been identified.³⁸ EK-O are also important for the synthesis of GA and can cause rice dwarfing.^68,69 Exogenously GA-treated RDV-infected plants exhibited restoration of phenotype of non-dwarf plants, but Aux treatments did not exhibit such results in this regard.³⁸ Virus-directed EK-O-mediated manipulations resulted in symptom development further leading towards the potential interference with the antimicrobial phytoalexin synthesis, thus making the plant more susceptible towards virus infections.⁴⁸ Viral component/proteins can cause the development of symptoms to cauliflower, e.g., Cauliflower mosaic virus (CaMV) P6 protein.⁷⁰ Furthermore, this multifunctional P6 viral protein plays a vital role in virus movement, replication, and RNAi suppression.^70–72 P6 transgenic expression induces the symptoms like stunting, chlorosis, and banding of veins in leaves.³⁹ Expression of P6 results in interference with the ET pathway’s response; for example, an ET-insensitive phenotype was displayed by P6 transgenic Arabidopsis plants.³⁹ It has been observed that P6 can cause inference with ET-signaling which may produce symptoms.⁷³ However, a direct relationship between P6 and various components of the ET pathway has not yet been identified.

Figure 1.

Role of phytohormones in plant–virus interactions. Here, some abbreviations include IAA (indole acetic acid), NRP1 (neuropilin-1), and MeJA (methyl jasmonate). BZR (brassinazole), (p)ppGPP (nucleotides guanosine tetraphosphate and pentaphosphate), ABA (abscisic acid), TuMV (Turnip mosaic virus), CaMV (Cauliflower mosaic virus), RDV (Rice dwarf virus), TMV (Tobacco mosaic virus), CMV (Cucumber mosaic virus), TsWV (Tomato spotted wilt virus), and RBSDV (Rice black-streaked dwarf virus). The articles cited include Refs. ^29–45.

3. Plant defense strategies and phytohormones

Several phytohormones play a crucial role in plant growth, development, and implementation of plant defense strategies against virus infections (Figure 2). For example, SA regulates basal defense via activating the mechanisms related to systemic acquired resistance (SAR) through R gene.^74,75 SA synthesis, activation, and signaling may result in accumulation of reactive oxygen species, callose deposition, activation of pathogenesis-related (PR) proteins, and induction of hypersensitive response.^76–78 For activation of SAR, SA is pre-requisite.⁷⁹ Antagonism has been observed for SA- and JA/ET-mediated defense-related pathways, particularly in case of viruses, insects, and necrotrophs.^80,81 However, synergism does exist between these pathways.⁸² In many studies, it has been explained that SA endogenous depletion or SA signaling disruption leads to defense response impairment and viral infection susceptibility.^83–86 For example, SA accumulation reduces salicylate hydrolase via using NahG transgene, further negating the resistance conferred against Potato virus Y (PV-Y) by the potato Ny-1 R-gene.⁸⁷ Thus, SA synthesis inhibition or SA-dependent defenses are one of the strategies which viruses may use for enhancing their infection. Proteins–virus interactions show its impact upon SA functions. For example, TMV replication protein ATAF2, which targets the degradation of NAC transcription factor domain’s proteasome and regulates plant basal defenses, disrupts SA functions.^40,88 Knockout of ATAF2 and SA treatment to repressor lines exhibited a reduction in the level of marker gene, PR1. Moreover, SA treatment does not reveal PR1 production in infected leaves, indicating that plant defense was attenuated because of systemic movement of TMV.⁸⁸ These results show that TMV-targeted ATAF2 degradation is responsible for the SA-mediated defense suppression. Second, it is proposed that CaMV P6 protein inhibits SA-dependent defense response with the expression, alternation, and localization of SA receptor NPR1.⁴¹ P6-expressing plants display a miss localization of an inactive form of NPR1 to the nucleus. This happens via disruption of the SA signaling pathway. It makes P6-expressing plants more resistant to JA but more susceptible to SA-sensitive pathogens, respectively.⁴¹ Additionally, current findings with TMV-Cg (a strain of crucifer) have shown that the virus coat protein (CgCP) may suppress signaling of SA by stabilizing of DELLA proteins without altering the SA or JA levels.⁷⁰ For GA signaling, DELLA proteins negatively regulate and repress the defense SA-mediated defense response, thus displaying antagonism between SA and JA pathways.^89,90 Additionally, expression of CgCP also reduces the growth of plants and delays floral transition timing.⁸⁹ Therefore, JA–SA antagonism is an important factor regarding plant defense against viruses.⁹¹ In N gene resistance embedding tobacco lines, exogenously applied methyl jasmonate may cause the reduction of TMV resistance.⁹² Additionally, silencing of JA receptor COI1 or a JA biosynthetic enzyme allene oxide synthase, resulted in enhanced SA production and reduced TMV accumulations in N gene embedded tobacco lines.⁹² JA–SA antagonism is also vital during gene expression-mediated defense. For example, RCY1 gene activates the resistance to Cucumber mosaic virus (CMV) via COI1 mutant allele (JA receptor) by minimizing the SA accumulation.^77,93 On the contrary, enhanced JA accumulation and its increased levels are not always favorable for plant defense against viral infections. It is reported that increased endogenous JA levels have been incompatible to plant–virus interactions in tobacco and potato.^94,95 Additionally, JA exogenous application disrupts infection of geminiviruses.⁹⁶ Furthermore, the C2 protein of the geminiviruses interacts with COP9 signalosome catalytic subunit, thus compromising the SCF activity of ubiquitin ligase and altering JA regulate ability.⁹⁷ SCF ubiquitination targets C2 proteins, which modulate plant resistance against geminiviruses through RNA silencing.⁶⁵

Figure 2.

Positive impact of phytohormones on plant defense mechanisms against virus infection. Salicylic acid (SA) signaling exhibits the major defensive pathways through nucleotide-binding leucine-rich repeat (NB-LRR) genes. These defense pathways are triggered by R proteins through SA activation, RNA interference, accumulation of reactive oxygen, and hypersensitive responses. These pathways activate the plant defenses against viral infection where systemically acquired resistance (SAR) and siRNAs are activated by SA at distal sites. Similar to SA, cytokeratins (CKs) and brassinosteroids (BRs) strengthen the plant defense against biotrophs.

BRs also activate plant defense against viruses.⁹⁸ Increased resistance against TMV was recorded upon the treatment of WT tobacco plants with brassinolide (BL). No SA accumulation or induction of PR gene expression was observed in BL-treated tobacco plants.⁹⁹ Furthermore, various geminiviruses exhibit interaction with the BR signaling pathway. For example, BRASSINOSTEROID-INSENSITIVE 2 (BIN2), a negative regulator of BR signaling, interacts with viral C4/AC4.¹⁰⁰ But the functional importance of this interaction requires further investigation. Correspondingly, Beat curly type virus (BCTV) C4 protein ectopic expression in Arabidopsis significantly alters plant development via disruption of multiple hormonal pathways.¹⁰¹ Moreover, the LRR-RLK brassinosteroid insensitive-1 (BRI1) that is a BR receptor and PRRs interact with the co-receptor [BRI1-associated kinase 1 (BAK1)] in a ligand-dependent manner. BAK1 is considered essential for plant defense against RNA viruses. For example, bak1-4 and bak1-5 mutants showed higher levels of Turnip crinkle virus (TCV), Oilseed rape mosaic virus (ORMV), and TMV as compared to WT plants.^102,103 The mutant plants are hypersensitive to ABA.¹⁰⁴ It is thought that enhanced AGO1 (a component of RNA silencing pathway) levels lead to hyposensitivity of ABA.^105,106 Additionally, miR168 (AGO1 regulator) contains elements of ABA-response in its promoter region and is upregulated by ABA.¹⁰⁷ There is also cross-talk evidence between SA and various pathways of defense through silencing mechanisms.^65,108 It was found that NahG expression by plants for reduced SA accumulations lowers the levels of small interfering RNAs (siRNAs) when infected with Plum pox virus (PPV).¹⁰⁹ Furthermore, potyvirus silencing suppressor protein HC-Pro overexpression reduced SA-mediated defenses against PPV.¹¹⁰ All these findings suggest that phytohormone signaling and virus defense responses have a strong connection.

4. Virus attacking strategies and phytohormones

Recent research findings demonstrated that phytohormones are directly and indirectly linked to the virus replication and their movement, thus indicating that virus-mediated alterations in hormone levels exhibit negative effects upon plant health (Figure 3). SA inhibits Tomato bushy stunt virus (TBSV) replication in one system by competitive bindings of cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH).¹¹¹ Cytosolic GAPDH binds the TBSV-negative RNA strand and has an important role in replication.¹¹² SA accumulations can lead towards its direct binding to GAPDH which actively prevents its interaction to RNA of virus paving the way for TBSV replication suppression.¹¹¹ Similarly, mutagenesis in ABA2 gene during biosynthesis of ABA resulted in reduction of Bamboo mosaic virus (BMV).^113,114 Similarly, failure in replication of CMV was recorded in aba2-1 mutant. This suggests that ABA2 is essential for virus replication or takes part in the translation of complex components.

Figure 3.

Negative impact of phytohormones upon plant defenses against viruses. Aux and SA have an antagonistic relationship. Movement of several types of viruses is dependent upon Auxin response factors (ARFs), e.g., TMV. Similarly, Et and SA show antagonism and are actively involved in CaMV symptom development. TMVcg also antagonizes the pathway downstream of SA signaling and is involved in symptom development on Cauliflower mosaic virus (CaMV) infection, systemic movement of TMVcg, and infection establishment. JA and ABA show multiple characteristics regarding plant defenses. For example, at earliest of the virus infection, JA supports plant defense systems, but once the infection is established solely and gets matured, JA production turns towards the favor of virus. ABA antagonizes with the SA pathway leading towards the reduction in resistance at local sites of infection.

Virus movement from cell-to-cell occurs through the vascular phloem via connections of intercellular plasmodesmata (PD).¹¹⁵ Mounting evidence indicates that in regulating PD connections, several phytohormones play an important role. For example, ABA hinders the movement of viruses through b-1,3-glucanase inhibition which degrades callose.^116–118 There is no doubt that callose deposition at PD decreases cell-to-cell viral movement.^119,120 Furthermore, ABA exogenous application increases the resistance of plants against viruses such as TMV and Tobacco necrosis virus (TNV) by hindering the virus movement.^121–123 Another phytohormone which is closely linked to the closure of PD is SA. Specifically, SA exogenous application results in the deposition of callose within PD.¹²⁴ This SA-mediated PD closing requires the activity of SA signaling pathways as well as PD gating regulator PDLP5.^125,126 SA also inhibits virus movement and replication via another process which requires the involvement of mitochondrial alternative oxidase (AOX) pathway.^127,128 Specifically, AOX functions as an inhibitor to counter SA-induced resistance against TMV, PVX, and CMV.

SA has the ability to control extreme resistance (ER), regulated by R genes. ER resembles effector-triggered immunity and completely eliminates the virus infection in the TBSV-resistant tobacco plants.¹²⁹ Similar examples include the soybean Rsv1-mediated resistance to Soybean mosaic virus (SMV)¹³⁰ and the Tm-22-mediated resistance against TMV or Tomato mosaic virus (ToMV).¹³¹ Sansregret et al. (2013) further observed that an intact SA pathway was required for TBSV resistance in tobacco plants. In tobacco, P19, which is a TBSV viral silencing suppressor (VSR), triggers ER. The consecutive P19 expression resulted in hypersensitive response-like (HR-like) necrosis. From this finding, it was hypothesized that the resistant tobacco plants may have the ability to sense small amounts of P19, which further leads towards the triggering of ER. However, the functionality of P19 due to VSR may result in accumulation of TBSV to the levels, which are sufficient for triggering HR. It suggests that the VSR function of P19 is necessary to induce HR. For example, the P19 mutants who lack in the VSR activity failed to induce HR.¹²⁹ On a similar note, Arabidopsis SA mutants npr1, sid2 (salicylic acid induction defi-cient2), eds5 (enhanced disease susceptible5), and pad4 (phytoalexin deficient 4) shown higher accumulation of CMV or ORMV as compared to the WT Col-0 plants, thus concluding that SA slowly triggers upon CMV or ORMV infection.¹³² Correspondingly, Wang et al.¹³³ compared the CP levels of CMV and TCV in eds5 and NahG mutants. The results were similar to previous findings up to 5dpi, but CPs were substantially increased in mutants at 10dpi followed by a gradual decrease after 15 dpi for both viruses. Similar trend of higher Bamboo mosaic virus (BaMV) RNA levels was also observed by Alazem et al.¹¹³ in the SA mutants eds1.

SA-mediated viral replication repression is thought to be correlated through siRNA pathway.^134,135 For example, P1/HC-Pro tobacco plants carrying NahG accumulated Tomato ringspot virus (ToRSV)-derived sRNAs in lesions.¹³⁶ More likely, SA treatment increased RNA-dependent RNA polymerase 1 (RDR1) levels in both tobacco and Arabidopsis.^122,137,138 However, genes encoding DCLs (dicer-like proteins) seem to be independent of SA-mediated resistance in Arabidopsis, as dcl2, dcl3, and dcl4 mutants exhibited that treating the plants with SA reduced CMV and TMV infection.¹³⁹ These results suggested that SA can be involved in triggering various DCL redundant mechanisms.¹³⁹ Interestingly, SA and ToMV infection induced DCL1, DCL2, RDR1, and RDR2 in tomato plants.¹⁴⁰ Some reports revealed that SA acts against VSRs. For example, NahG transgenic plants showed higher levels of CMV-2b virus (lacking the CMV2b suppressor) than WT plants.¹⁴¹ Moreover, there are pieces of evidence about strong linkage between siRNA pathways and SA biosynthesis. SA pathways are interfered by viral VSRs. For example, CMV-2b VSR directly affected SA regulations, further reducing the viral symptoms in N. glutinosa.^42,142,143

Recently, it is determined that TMV disrupts the selected phloem expressing Aux/IAA gene nuclear localization.⁵² This vascular-specific IAA regulatory protein disruption was found to be in correlation with the virus ability to load into the phloem and move systemically.¹⁴⁴ Therefore, TMV targets the pathway of Aux signaling, thus enhancing its systemic spread and ability to cause disease. Viruses can also move plant-to-plant by many insect vectors which represents another virus biology aspect conferred by phytohormones.^145,146 Particularly in plant–insect defenses, JA has been found as a primary hormone responsible for vector transmission. For example, western flower thrips which are vectors for Tomato spotted wilt virus (TSWV) prefer to feed on infected tissues.^147,148 SA production is increased, while JA levels are reduced in TSWV-infected plants.¹⁴⁹ However, it is still undisclosed whether these pathways are directly targeted by TSWV to enhance insect-mediated virus transmission. Furthermore, CMV-2b silencing suppressor interrupts JA signaling for enhancing vector-(Myzus persicae)-mediated transmission. Moreover, CMV-infected plants or other transgenics expressing 2b exhibited the least levels of JA.⁴³ Additionally, it is interesting to note that aphid survival on CMV-infected tobacco plants (carrying 2b) increases. Oppositely, the survival of aphids decreases when they feed upon the tobacco plants lacking 2b.⁹⁴ Finally, TuMV NIa-Pro (Nuclear Inclusion–Protease domain) alters the responses of ethylene-suppressing aphid-induced callose defenses. Among potyviruses, NIa-Pro is highly conserved, and researchers propose a conserved mechanism of this interaction for transmission of viruses through aphids.¹⁵⁰

5. Concluding remarks

Phytohormones are significant in numerous aspects of plant–virus interactions. Phytohormones have multiple linkages to changes occurred in the accumulation of viruses. It has also been found that cross talk between the pathways of phytohormones is critical for virus defense response regulation. However, despite technological advances in the twenty-first century, we still lack specific information regarding genes that regulate phytohormones and pathways that affect virus biology. Which Aux-regulated genes are responsible for the development of symptoms or which SA-mediated processes impact directly on the accumulation of virus are just one of the few questions that are still unanswered. The answers to these questions will provide a better understanding of the involvement of phytohormones in plant–virus interactions. Furthermore, we should focus on molecular mechanisms that how viruses target and modulate the synthesis of plant hormone and sensing systems to avoid plant defenses, thus enhancing their movement and infection ability.