Front. Plant Sci. Frontiers in Plant Science Front. Plant Sci. 1664-462X Frontiers Media S.A. 10.3389/fpls.2022.1083409 Plant Science Review TIR1/AFB proteins: Active players in abiotic and biotic stress signaling Du Wenchao 1 † Lu Yang 2 † Li Qiang 1 Luo Shuangxia 1 Shen Shuxing 1 Li Na 1 * Chen Xueping 1 * 1 Key Laboratory for Vegetable Germplasm Enhancement and Utilization of Hebei, Collaborative Innovation Center of Vegetable Industry in Hebei, College of Horticulture, Hebei Agricultural University, Baoding, China 2 Hebei University Characteristic sericulture Application Technology Research and Development Center, Institute of Sericulture, Chengde Medical University, Chengde, China

Edited by: Chao Li, Northwest A&F University, China

Reviewed by: Zhilong Bao, Shandong Agricultural University, China; Dongfeng Jia, Jiangxi Agricultural University, China

*Correspondence: Na Li, yyln@hebau.edu.cn; Xueping Chen, chenxueping@hebau.edu.cn

†These authors have contributed equally to this work

This article was submitted to Plant Abiotic Stress, a section of the journal Frontiers in Plant Science

 29 11 2022 2022 13 1083409 29 10 2022 16 11 2022 Copyright © 2022 Du, Lu, Li, Luo, Shen, Li and Chen 2022 Du, Lu, Li, Luo, Shen, Li and Chen

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The TIR1/AFB family of proteins is a group of functionally diverse auxin receptors that are only found in plants. TIR1/AFB family members are characterized by a conserved N-terminal F-box domain followed by 18 leucine-rich repeats. In the past few decades, extensive research has been conducted on the role of these proteins in regulating plant development, metabolism, and responses to abiotic and biotic stress. In this review, we focus on TIR1/AFB proteins that play crucial roles in plant responses to diverse abiotic and biotic stress. We highlight studies that have shed light on the mechanisms by which TIR1/AFB proteins are regulated at the transcriptional and post-transcriptional as well as the downstream in abiotic or biotic stress pathways regulated by the TIR1/AFB family.

 TIR1/AFB abiotic stress biotic stress structural and functional specialization transcription National Natural Science Foundation of China10.13039/501100001809 Introduction

Transport Inhibitor Response 1 and Auxin-Signaling F-box (TIR1/AFB) proteins are plant-specific receptors that mediate diverse responses to the plant hormone auxin (Dharmasiri et al., 2005; Parry et al., 2009). Upon binding indole-3-acetic acid (IAA), or other hormones in the auxin class, TIR1/AFB proteins form a co-receptor complex with Auxin/IAA (Aux/IAA) proteins (Salehin et al., 2015). Formation of this co-receptor complex results in ubiquitination and degradation of Aux/IAA proteins via the 26S proteasome (Pan et al., 2009; Salehin et al., 2015; Todd et al., 2020). Degradation of Aux/IAA proteins releases their inhibition of auxin response factors (ARFs), which are transcriptional regulators of auxin-responsive genes such as Aux/IAA (Strader and Zhao, 2016; Yu et al., 2022). In this way, TIR1/AFB proteins serve as positive regulators of downstream auxin-responsive pathways upon the perception of auxin (Quint and Gray, 2006; Dezfulian et al., 2016; Takato et al., 2017).

The first TIR1/AFB gene identified and shown to play an important role in auxin-regulated processes, such as hypocotyl elongation and lateral root formation, was TIR1 in Arabidopsis (Ruegger et al., 1998). Subsequent studies identified TIR1/AFB family members encoded in the genomes of algae, mosses, and spermatophytes in addition to all land plants (Parry et al., 2009). The large number of TIR1/AFB genes encoded in plant genomes has allowed for functional redundancy and neofunctionalization to evolve (Prigge et al., 2020). It is now clear that TIR1/AFB proteins contribute to biological processes including regulation of primary and secondary metabolism (Gomes and Scortecci, 2021), seed and root development (Pan et al., 2009; Ozga et al., 2022), cell proliferation (Rast-Somssich et al., 2017), immunity and stress responses in plants (Iglesias et al., 2010). In this review, we highlight our current understanding of the structure and function of TIR1/AFB family members with an emphasis on possible mechanisms by which these proteins regulate abiotic and biotic stress responses.

 Structural and functional specialization of TIR1/AFB family members in <italic>Arabidopsis</italic>

Based on comparisons of land plant genomes sequenced to-date, TIR1/AFB proteins can be divided into four phylogenetic clades: TIR1/AFB1, AFB2/3, AFB4/5, and AFB6. Arabidopsis contains six TIR1/AFB proteins from three out of the four clades: TIR1, AFB1, AFB2, AFB3, AFB4, and AFB5 (Shimizu-Mitao and Kakimoto, 2014). AFB6 orthologs are noticeably absent in the core Brassicales species such as Arabidopsis as well as Poaceae species such as rice and maize (Prigge et al., 2020).

The specific functions of TIR1/AFB family members vary considerably across and within clades. For instance, AFB4 and AFB5 are in the same clade yet exhibit distinct specificities for auxin (Prigge et al., 2016). Yeast two-hybrid and immunoblot assays demonstrated that IAA3 binds TIR1, AFB1, and AFB2 with different affinities but binds AFB5 very poorly at 0.1 μM IAA. Distinct motifs are necessary for the assembly of TIR1/AFB-IAA coreceptor complexes (Villalobos et al., 2012). Here, we generated a phylogenetic tree containing all TIR1/AFB family members from Arabidopsis and used Motif ENRichment Analysis (MEME) to identify conserved protein motifs ( Figure 1 ). We believe the unique motifs present in TIR1/AFB proteins may explain their preferential binding of certain IAA proteins over others.

Neighbor-joining phylogenetic tree (left) and conserved motif (right) analysis of TIR1/AFBs in Arabidopsis.

Our analysis showed that Arabidopsis TIR1/AFB proteins contain different conserved motifs. These proteins consist of a single F-box domain and eighteen LRR repeats (Prigge et al., 2020). F-box domains are critical for the regulated degradation of cellular proteins (Jain et al., 2007) while LRRs belong to an archaic procaryal protein architecture that is widely involved in protein-protein interactions (Martin et al., 2020). We found that different TIR1/AFB family members contain unique motifs. Motifs 1 and 12 are only present in AFB4 and AFB5, motifs 11 and 20 are only present in AFB2 and AFB3, motif 14 is only present in AFB3, and motif 9 is only found in AFB4. The presence and absence of certain motifs indicates that TIR1/AFBs may have different functions.

Synthetic auxin herbicides are one of the most potent man-made abiotic stresses that plants are subjected to (Gorina et al., 2022). Picloram, 2,4-dichlorophenoxy acetic acid (2,4-D), and dicamba are three of the most widely used chemical classes of auxin. These herbicides function by binding to a hydrophobic pocket within TIR1/AFB proteins (Meng et al., 2008; Guo et al., 2021). Auxin binding TIR1 by filling in the bottom of TIR1 pocket, which floor is made up of several key residues containing His 78, Arg 403, Ser 438, Ser 462, and Glu 487 as shown in ( Figure 2 ) (Guo et al., 2021). Distinct amino acid residues exist in the AFB4/5 clade compared with the TIR1/AFB1 and AFB2/3 clades at His 78 and Ser 438: histidine is replaced by arginine and serine is replaced by alanine. These differences demonstrate the diversity of TIR1/AFB members and suggest a structural reason for their specialized responses to different synthetic auxin herbicides.

Multiple alignment of TIR1/AFB proteins in Arabidopsis adapted from Fu Guo et al. (Guo et al., 2021). Residues highlighted in gray are present in more than 50% of aligned sequences. The key residues making up the active site of the TIR1 pocket are highlighted by red boxes. Accession numbers of the genes encoding the proteins for the sequence alignment are as follows: TIR1 (At3g62980), AFB1 (At4g03190), AFB2 (At3g26810), AFB3 (At1g12820), AFB4 (At4g24390), and AFB5 (At5g49980).

Studies on Arabidopsis mutants have demonstrated that some members of the TIR1/AFB family are responsible for the recognition of specific auxin herbicides (Grossmann et al., 1996). For instance, the Arabidopsis afb4/5 mutant is resistant to picloram whereas other tir1/afb mutants are still susceptible (Walsh et al., 2006). The AFB4 protein itself was shown to be a target of picloram based on in vitro binding assays (Prigge et al., 2016). TIR1 has been shown to be a receptor for 2,4-D and induces changes in gene expression when plants are treated with low concentrations of 2,4-D (Sheedy et al., 2006; Walsh et al., 2006). As anticipated, the Arabidopsis tir1 mutant is resistant to 2,4-D whereas AFB1, a member of the same clade as TIR1, has not been implicated in 2,4-D resistance (Gleason et al., 2011).

In vitro assays demonstrated that TIR1 and AFB5 can bind to dicamba (de Figueiredo et al., 2022). Of all the TIR1/AFB family members in Arabidopsis, only the tir1-1 and afb5 mutants were shown to be resistant to dicamba (Gleason et al., 2011). No studies have yet implicated the AFB2/3 subgroup in auxin herbicide sensitivity, which further demonstrates the structural and functional specialization that exists in the TIR1/AFB family. However, studies on the rice mutants Osabf2 and Osabf3 showed OsAFB2/3 genes are involved in the response to 2,4-D resistance (Guo et al., 2021). These results suggest that more studies should focus on the function of the AFB2/3 subgroup in herbicide susceptibility.

 The role of TIR1/AFB family members in abiotic and biotic stress responses

Plants are sessile organisms challenged by a variety of abiotic and biotic stresses from which they cannot escape. Abiotic stresses are caused by environmental conditions such as drought, high salinity, heat, and cold whereas biotic stresses are caused by living organisms such as bacteria, fungi, viruses, nematodes, and insects (Verma et al., 2016; Burns et al., 2018). Both abiotic and biotic stress induce reactive oxygen species (ROS) production in the form of hydroxyl radicals, hydrogen peroxide, and superoxide anions (Singh et al., 2020). At low concentrations, many ROS species function as signaling molecules in stress tolerance pathways. However, elevated and sustained levels of ROS can become toxic and lead to nutrient loss, resulting in metabolic disruption, abnormal hormone metabolism (Rejeb et al., 2014; Muchate et al., 2016), and growth inhibition (Gimenez et al., 2018). Auxin plays an indispensable role in how plants rapidly adapt to abiotic and biotic stress. As key auxin receptors in plants, the TIR1/AFB family has been shown to be essential for abiotic and biotic stress responses mediated by auxin.

Drought stress

Drought is an important abiotic stress that negatively impacts plant development and results in reduced crop yield and quality. The expression of many TIR1/AFB genes is influenced by drought stress, which suggests the TIR1/AFB family may function in the drought tolerance pathway (Shu et al., 2015; Sharma et al., 2018; Benny et al., 2019). Over-expression and transcriptomic studies in Populus trichocarpa, Arabidopsis thaliana, Oryza sativa, Zea mays, Solanum tuberosum, Triticum aestivum, and Agrostis stolonifera have demonstrated that many TIR1/AFB genes are responsive to drought (Chen et al., 2012; Shu et al., 2015; Dalal et al., 2018; Sharma et al., 2018; Benny et al., 2019; Zhao et al., 2019). Relative water content (RWC) is used as a measure of plant water status and is a meaningful index of water stress tolerance (Lo Gullo and Salleo, 1988). PtrFBL1 is a TIR1 homolog in Populus trichocarpa. Overexpression of PtrFBL1 in P. trichocarpa resulted in higher plant RWC values upon drought stress compared with non-transgenic plants (Shu et al., 2015).

Gene expression analyses suggest that some TIR1/AFB family members participate in drought responses in Arabidopsis. For example, TIR1 and AFB2 are required for the inhibition of lateral root growth by ABA or osmotic stress under drought stress (Chen et al., 2012). In seedling studies, TIR1 was up-regulated under drought stress as determined by RNA-Seq (Benny et al., 2019). In addition to the well-studied Arabidopsis TIR1/AFB family, several TIR1/AFB proteins have also been implicated in drought responses in other species by transcriptional analysis. In rice, TIR1 and AFB2 expression levels were significantly downregulated in spikelets upon drought stress (Sharma et al., 2018). In maize and the Solanaceous crops tomato and potato, RNA-Seq results demonstrated that TIR1 expression increased in seedlings exposed to drought stress (Benny et al., 2019). Drought-stressed roots of the wheat genotype viz. Raj3765 had increased expression of AFB2, suggesting AFB2 may play a key role in response to drought (Dalal et al., 2018). Creeping bentgrass (Agrostis stolonifera L.) overexpressing the rice pri-miR393a exhibited improved tolerance to drought stress due to targeting and suppression of AsAFB2 and AsTIR1 expression (Zhao et al., 2019).

 Salt stress

Salt stress is a major environmental factor limiting plant growth and productivity. Salt stress can lead to ionic stress, osmotic stress, and secondary stresses such as oxidative stress (Yang and Guo, 2018). Mutant, overexpression, and ectopic expression studies of TIR1/AFB genes in Arabidopsis have uncovered a key role for some of these genes in salt stress tolerance. Expression of AtNAC2, which is typically induced by salt stress, is unresponsive to salt stress in the tir1-1 mutant (He et al., 2005). An Arabidopsis tir1afb2 double mutant exhibited enhanced tolerance against salt stress compared with wild-type plants as determined by a higher germination rate, greater root elongation, and higher chlorophyll content (Iglesias et al., 2010). The cucumber (Cucumis sativus L.) CsTIR1 and CsAFB1 proteins share 78% and 76% amino acid identity with their Arabidopsis homologs, respectively. However, ectopic overexpression of CsTIR1 and CsAFB1 in Arabidopsis led to higher germination and plant survival rates under salt stress (Chen et al., 2017). Over-expression of the Arabidopsis AFB3 in Arabidopsis resulted in better primary and lateral root development and higher germination rates upon salt stress compared with the wild type (Garrido-Vargas et al., 2020).

It certainly seems contradictory that a tir1afb2 double mutant and overexpression of AFB3 or CsTIR1/CsAFB1 both enhance salt stress resistance in Arabidopsis. This may be explained by increased activity of antioxidant enzymes in the tir1afb2 mutant under salt stress. Higher levels of ABA are also detected in tir1afb2 compared with wild-type plants (Iglesias et al., 2010) while more lateral roots are found in Arabidopsis transgenic lines overexpressing AFB3, CsTIR1, or CsAFB1 (Chen et al., 2017; Garrido-Vargas et al., 2020). This may contribute to differential participation of TIR1/AFB family members and their tissue-specific functions (Iglesias et al., 2010; Garrido-Vargas et al., 2020).

In addition to numerous studies in Arabidopsis, TIR1/AFB proteins have also been implicated in salt stress responses in other plant species. Overexpression of maize ZmAFB2 in tobacco led to enhanced salt tolerance (Yang et al., 2013). Eighteen TIR1/AFB genes have been identified in Brassica juncea var. tumida with qPCR analysis, which showed that some BjuTIR1/AFB genes are repressed by salt treatment (Cai et al., 2019). Degradome and miRNA sequencing analysis between salt-tolerant and salt-sensitive Fraxinus velutina Torr. tree cuttings demonstrated that reduced expression of TIR1 by miR393a explains the enhanced salt stress tolerance of this tree species (Liu et al., 2022). Interestingly, AsAFB2 and AsTIR1 from creeping bentgrass may serve as a link between drought and salt stress response pathways, both pathways rely on ionic and osmotic homeostasis signaling (Zhu, 2002; Zhao et al., 2019), and AsAFB2 and AsTIR1 have been implicated involving in this process (Zhao et al., 2019). It is thus plausible that some TIR1/AFB family members may serve as key regulators of plant responses to multiple abiotic stresses.

 Temperature stress

Temperature is one of the most important environmental signals for plants. High and low temperatures have a variety of effects that affect plant growth and development profoundly (Sakamoto and Kimura, 2018). Expression data from different plant species indicates that members of the TIR1/AFB family participate in plant responses to temperature stress. For example, the Arabidopsis tir1-1 mutant displays defective hypocotyl elongation at elevated temperatures (Gray et al., 2003). Expression of TIR1/AFB2 in rice spikelets was significantly downregulated by heat stress, and the rice protein OsAFB6 can suppress flowering, which is thought to be a temperature sensor (He et al., 2018; Sharma et al., 2018). Finally, repression of TIR1 expression in wheat impairs pollen exine formation in male sterility under cold stress (Liu et al., 2022).

 Phosphorus and nitrate stress

Phosphorus (Pi) and nitrate (a main source of inorganic nitrogen) are crucial nutrients for crop growth and development that are mainly absorbed from soil by roots. Phosphorous deficiency and excessive nitrate result in retardation of plant growth, development, and productivity (Koide et al., 1999; Zhang et al., 2017). The first TIR1/AFB protein found to be involved in Pi and nitrate availability is TIR1 from Arabidopsis, which was shown to be involved in pattern alterations of lateral root formation and emergence in response to phosphate availability (Perez-Torres et al., 2008). The expression level of TIR1 is also induced under low Pi conditions (Mayzlish-Gat et al., 2012).

Regulation of root system architecture by external nitrate is mediated by AFB3 in Arabidopsis as demonstrated by afb3 insertional mutants (Vidal et al., 2010). Integrated genomics, bioinformatics, and molecular genetics revealed that the expression of genes downstream of AFB3 are influenced by external nitrate with the NAC4 transcription factor serving as a key regulator of this network (Vidal et al., 2013). AFB3-mediated activation of the two independent pathways in response to nitrate suggests that AFB3 is a unique nitrate response factor in Arabidopsis (Vidal et al., 2010). TIR1/AFB family members were also found to be key players in response to nitrate in other plant species. In Lotus japonicus, expression of LjAFB6 is induced in response to exogenous nitrate (Rogato et al., 2021). These studies indicate that AFB3 in Arabidopsis and LjAFB6 in L. japonicus are potentially involved in plant responses to stress caused by excessive nitrate.

 Herbicide stress

Herbicides are small molecules that inhibit specific molecular target sites within plant biochemical pathways to affect physiological processes. Inhibition of these sites often has catastrophic consequences that are lethal to the plant (Dayan et al., 2010). Synthetic auxin, triazine, and organophosphorus herbicides are commonly used in agriculture to control weeds (Todd et al., 2020; Bigner et al., 2021; Striegel et al., 2021). Multiple members of the TIR1/AFB family are involved in susceptibility to synthetic auxin herbicides. Studies on Arabidopsis TIR1/AFB mutants have revealed a role for these genes in response to classical auxin herbicides. Recently, the afb5 mutant was found to be resistant to a new auxin herbicide, halauxifen-methyl, which preferentially binds to AFB5 (Xu et al., 2022).

TIR1/AFB proteins also play a key role in the response to auxin herbicides in other plant species. In rice, CRISPR/Cas9 genome editing was used to generate Ostir1/Osafb2/Osafb3/Osafb4/Osafb5 mutants that was resistant to 2,4-D. Osafb4 mutants are highly resistant to the herbicide picloram (Guo et al., 2021). Expression of TIR1 in wheat is clearly higher in Triticum aestivum than in Aegilops tauschii, resulting in less sensitive to the herbicide 2,4-D (Yu et al., 2021).

 Emerging evidence implicates TIR1/AFB proteins in metal stress tolerance and boron deficiency

In addition to the stresses described above, emerging evidence suggests that TIR1/AFB proteins may be involved in plant responses to metal, and boron deficiency. Aluminum toxicity inhibits plant growth and development (Liu et al., 2022). Inhibition of root morphogenesis under aluminum stress decreased in Arabidopsis tir1 single and tir1 afb2 afb3 triple mutants. Other genes in the auxin signaling pathway, such as ARFs, were also shown to be involved in aluminum sensitivity (Ruiz-Herrera and Lopez-Bucio, 2013). MicroRNAs targeting and mediating the cleavage of TIR1/AFB transcripts were shown to be essential for the aluminum stress response in Arabidopsis (Mendoza-Soto et al., 2012). These results suggest TIR1, AFB2, AFB3, and downstream auxin-responsive genes play an important role in aluminum sensitivity in Arabidopsis.

Boron is an abundant and essential micronutrient required by plants with deficiencies causing impaired plant growth (Park et al., 2005; Duran et al., 2018). Boron deficiency is positively correlated with the expression of many miRNAs. Gene expression analysis indicates that a subgroup of miRNAs regulate TIR1/AFB expression in Arabidopsis when boron is limited. This leads to decreased expression of TIR1, AFB1, and AFB2 but increased expression of AFB3 (Lu et al., 2015). Other reports have demonstrated that application of α-(phenylethyl-2-oxo)-indole-3-acetic acid (PEO-IAA), a synthetic antagonist of TIR1, could partially or fully restore cell elongation in boron deficient roots (Camacho-Cristobal et al., 2015).

 Biotic stress from pathogenic bacteria, fungi, viruses, nematodes, and phytophagous insects

Biotic stresses are mainly caused by pathogenic species of bacteria, fungi, viruses, nematodes, and insects that seek to acquire nutrients from their plant hosts (Jagdale and Joshi, 2019; Bhar et al., 2022). Damages caused by diseases and herbivory reduce crop yield and quality by affecting photosynthesis and secondary metabolite production in the host plant (Vo et al., 2021). Plants have evolved numerous strategies to defend themselves against these pathogens. These strategies rely on coordinated gene, protein, and hormone regulation to allow plants to sense and adapt to biotic stresses (Atkinson and Urwin, 2012). Auxin is a critical signaling component of the plant response to biotic stress, which suggests that TIR1/AFB proteins have a role to play as well (Ghanashyam and Jain, 2009; Bouzroud et al., 2018; Gidhi et al., 2022).

Plant pathogenic bacteria cause symptoms such as spots with yellow halos or mucus-like materials, which negatively impact agricultural production in many important crops (Zimaro et al., 2011). The tomato bacterial pathogen Pseudomonas syringae DC3000 (PtoDC3000) produces IAA to promote PtoDC3000 growth in plant tissues through suppression of SA-mediated host defenses (Wildermuth et al., 2001; McClerklin et al., 2018; Djami-Tchatchou et al., 2020). An Arabidopsis tir1afb1 afb4 afb5 quadruple-mutant exhibited elevated IAA levels and reduced SA levels compared with WT (Djami-Tchatchou et al., 2020). An analysis of a tir1 single mutant and tir1 afb2 afb3 triple mutant revealed that these TIR1/AFB family members are targeted by diketopiperazines derived from Pseudomonas aeruginosa during colonization of Arabidopsis (Ortiz-Castro et al., 2011). The planar structure of diketopiperazines likely fits into the same pocket of TIR1 that synthetic auxins bind (Ortiz-Castro et al., 2011).

Fungal plant pathogens are ubiquitous, highly diverse, and can cause severe damage to many important crops (Termorshuizen, 2016). The Arabidopsis afb1 and afb3 mutants are partially resistant to the soilborne root pathogen Verticillium dahlia. Up-regulation of pathogen-related gene 1 (PR1) in afb1 and pathogen defense factor 1.2 (PDF1.2) in afb3 may be responsible for afb1- and afb3-mediated resistance, respectively (Fousia et al., 2018). Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum Schwabe, results in large annual yield losses in wheat production regions. RNAi-mediated knockdown of the TaTIR1 gene led to increased FHB resistance (Su et al., 2021). Gene expression studies also revealed that TaTIR1 expression is highest at 24 and 48 h post-inoculation with the leaf rust pathogen Puccinia triticina Eriks (Gidhi et al., 2022). A maize TIR1-like gene is involved in the Zma-miR393b-mediated response to Rhizoctonia solani infection of leaf sheaths (Luo et al., 2014). Eighteen TIR1/AFB genes have been identified in Brassica juncea var. tumida using genome-wide analysis. qPCR analysis demonstrated that the expression of some BjuTIR1/AFB genes is influenced by Plasmodiophora brassicae infection (Cai et al., 2019).

Although no involvement in biotic stress has been reported for soybean TIR1/AFB proteins, TIR1/AFB proteins have been implicated in root nodulation induced by the nitrogen-fixing bacterium Bradyrhizobium japonicum (Cai et al., 2017). Overexpression of GmTIR1 in soybean significantly increased the number of inflection foci and nodules while GmAFB3A may also play a minor role in this process (Cai et al., 2017).

Few studies to-date have implicated the TIR1/AFB family in plant defense responses against viruses. However, one study has shown that the rice dwarf virus (RDV) capsid protein P2 binds OsIAA10 and blocks the interaction between OsIAA10 and OsTIR1. This prevents 26S proteasome-mediated degradation of OsIAA10, resulting in plant dwarfism, increased tiller number, and short crown roots in infected plants (Jin et al., 2016).

Nematodes are pathogens of Arabidopsis (Moradi et al., 2021), apple (Fallahi et al., 1998), tomato (Khan and Khan, 1995), and wheat (Cortese et al., 2003), these species could move through roots and be vector of some virus, caused root damage, yield loss. The tomato Mi-1 gene confers isolate-specific resistance against root-knot nematodes (Seah et al., 2007). Co-localization of TIR1-like proteins with the Mi-1 protein was observed (Seifi et al., 2011). TIR1-like transcript abundance in roots and leaves of nematode-resistant tomato lines was lower than in susceptible tomato lines, suggesting a possible role for TIR1-like genes in nematode resistance (Seifi et al., 2011).

Feeding by phytophagous insects such as aphids leads to reduced plant growth, reduced yield, water stress, dwarfism, wilting, and transmission of economically important plant viruses. In melon, genes like TIR1 and AFB2 are down-regulated in response to aphid herbivory. Application of the TIR1 inhibitor PEO-IAA to leaf discs resulted in significantly decreased feeding by aphids, providing in vivo support for TIR1/AFB in response to aphids (Sattar et al., 2016), suggested that TIR1 may play a role in aphid resistance.

 TIR1/AFB-regulated gene networks in abiotic and biotic stress responses

In addition to the regulation of Aux/IAA genes, many other proteins and genes regulated by TIR1/AFB family members have been identified that act downstream of auxin perception. These studies have contributed to our understanding of the mechanisms underlying the function of TIR1/AFB proteins in abiotic and biotic stress. These downstream genes and proteins include nascent polypeptide-associated complex (NAC) family members, SA synthesis proteins, PR proteins, PDF proteins and phosphorus transporters,

Auxin/indoleacetic acid (Aux/IAA) proteins play an important regulatory role in plant development and stress responses. TIR1/AFB proteins are essential regulators of the expression of a large number of Aux/IAA genes (Gray, 2003). For example, the rice Aux/IAA protein OsIAA20 mediates abiotic stress tolerance in rice through the ABA pathway (Zhang et al., 2021). Constitutive expression of OsIAA18 in Arabidopsis led to improved salt and osmotic tolerance through enhanced ABA biosynthesis and ROS scavenging (Wang et al., 2021). The homeostatic expression of Aux/IAA is thought to be one of the most important resistance mechanisms to auxin herbicides mediated by TIR1/AFB proteins (Todd et al., 2020).

Aux/IAA proteins also play essential roles in response to biotic stress. Silencing of GhIAA43 in cotton enhanced wilt resistance and activated the expression of SA-related defense genes (Su et al., 2022). Tobacco mosaic virus (TMV) replicase proteins negatively regulate IAA26 through a ubiquitin-mediated destabilization process to reduce TMV infection (Padmanabhan et al., 2005). The RDV capsid protein P2 can bind OsIAA10 directly, which implicates OsIAA10 in the defense response against RDV (Jin et al., 2016).

In addition to the Aux/IAA genes, many other stress-related genes are also regulated by TIR1/AFB proteins in response to abiotic and biotic stress. For example, the transcription factor NAC4 is an important positive regulator downstream of the AFB3 regulatory network, which plays an important role in the regulation of nitrate uptake in Arabidopsis (Vidal et al., 2013). The presence of a functional copy of NAC1 is required by the fungal pathogen Alternaria alternata for full virulence in Arabidopsis (Wang et al., 2020). NAC1 overexpression can restore lateral root formation in the Arabidopsis tir1 mutant, whereas TIR1 overexpression results in increased NAC1 expression. These results demonstrate that NAC1 acts downstream of and can be positively regulated by TIR1 in Arabidopsis (Xie et al., 2000).

The SA-related genes PR1 and PDF1.2 are positive regulators of plant disease resistance that are negatively regulated by TIR1/AFB. A transcriptomic study in cotton demonstrated that knockdown of GhTIR1 leads to a significant increase in the expression of SA-related genes in response to Verticillium dahliae infection (Shi et al., 2022). The Arabidopsis mutants afb1 and afb3 exhibit significantly higher expression of both PR1 and PDF1.2 in response to Verticillium dahliae infection (Fousia et al., 2018).

TIR1/AFB proteins act as mediators of low Pi uptake in Arabidopsis (Perez-Torres et al., 2008; Perez Torres et al., 2009). Pi deprivation increases the expression of TIR1 in Arabidopsis seedlings (Perez-Torres et al., 2008). ARF was regulated by TIR1/AFB as described above. Knockout of OsARF12 enhanced the expression of PHOSPHATE TRANSPORTER1(PHT1) genes such as OsPHR2 in rice, suggesting that OsARF negatively regulates the PHT1 gene family in rice (Wang et al., 2014).

 Regulation of TIR1/AFB expression and protein activity in response to abiotic and biotic stress

Many TIR1/AFB genes are differentially expressed in response to diverse abiotic or biotic stresses. Yet the underlying mechanism of TIR1/AFB gene regulation remains unknown.

TIR1 expression is up-regulated or down-regulated in Arabidopsis upon infection by plant pathogens such as Verticillium dahlia and Botrytis cinerea (Llorente et al., 2008; Fousia et al., 2018). Many plant pathogens manipulate host auxin biosynthesis, inducing the degradation of AUX/IAA proteins through TIR1-mediated ubiquitination to enable greater infection (Wang et al., 2007). The Arabidopsis mutants afb1 and afb3 have enhanced plant resistance against Verticillium dahlia. However, the tir1-1 mutant exhibits no increase in susceptibility to Botrytis cinerea compared to wild-type Arabidopsis. These studies indicate that TIR1/AFBs may be targeted by some pathogens.

Plant-produced small molecules are key systemic modulators of numerous biological pathways. Nitric oxide (NO) is an important signaling molecule involved in establishing resistance to plant stress. External NO represses TIR1 expression and decreases Arabidopsis susceptibility to Pseudomonas. syringae pv. tomato: a process believed to be mediated by SA (Vitor et al., 2013). Hydrogen sulfide (H2S) is a gaseous molecule involved in various responses to stress. H2S negatively regulates the expression of TIR1, AFB1, AFB2, and AFB3 in antibacterial resistance in Arabidopsis through a miR393a/b-regulated mechanism (Shi et al., 2015).

While most abiotic and biotic stresses suppress the expression of TIR1/AFB family members, some stresses can induce their expression. In L. japonicus, LjAFB6 expression increased by 2.5-fold after nitrate treatment (Rogato et al., 2021). Arabidopsis AFB3 was also found to be positively regulated by nitrate addition (Vidal et al., 2010; Vidal et al., 2013). Infections of Plasmodiophora brassicae in Brassica juncea var. tumida also induce the expression of BjuTIR1/AFB and BjuTIR1 (Cai et al., 2019), but the mechanism by which this process occurs is not yet clear.

Some members of the TIR1/AFB family involved in abiotic or biotic stress responses are known targets of small RNAs. One of the most well-studied small RNAs shown to target and repress TIR1/AFB transcripts is MicroRNA393 (miR393) (Navarro et al., 2006). In Arabidopsis, miR393 directly targets TIR1, AFB1, AFB2, and AFB2 transcripts in response to abiotic stress (Vidal et al., 2010; Chen et al., 2012; Iglesias et al., 2014). Regulation of AFB3 by miR393 represents a unique nitrate-responsive module that is induced by nitrate and repressed by nitrogen metabolites in Arabidopsis (Vidal et al., 2010). Studies also indicate that miR393 negatively regulates TIR1, AFB2, and AFB3 in response to pathogen challenge in several plant species (Navarro et al., 2006; Zhang et al., 2019; Shi et al., 2022). Though studies indicate that miR393 negatively regulates TIR1 expression at the posttranscriptional level (Parry et al., 2009), the relationship between miR393 and TIR1/AFB transcripts needs to be investigated further.

In addition to regulated gene expression or posttranscriptional level, TIR1/AFB proteins are also regulated post-translationally by other proteins. The Arabidopsis TIR1 protein is stabilized by a complex consisting of heat shock protein 90 (HSP90) and Suppressor of G2 allele of skp1 (SGT1b), which itself is an HSP90 co‐chaperone, co-immunoprecipitation analyses further validated that HSP90 interacted with TIR1 (Watanabe et al., 2016; Munoz et al., 2022). So far, no other factors were found to positively or negatively regulate TIR1/AFB proteins at post-translational level under stress. Therefore, future study should explore factors that regulate or interact with TIR1/AFB proteins.

 Conclusions and perspectives

Phylogenetic, structural, and functional studies have revealed that there are many homologs of TIR1/AFB proteins with conserved domains. Many TIR1/AFB genes are differentially expressed in response to diverse abiotic and biotic stress ( Table 1 ). Small molecules such as NO and H2S regulate TIR1/AFB gene expression, MicroRNAs, such as miR393, are some of the most well-studied regulators of TIR1/AFB transcripts. The regulation of some TIR1/AFB family members through protein-protein interactions and small molecules is also indispensable ( Figure 3 ). Future studies should focus on identifying more factors that can regulate TIR1/AFB family members at the transcriptional, post-transcriptional, and protein levels. These studies will shed light on the evolution of the TIR1/AFB family and identify new roles for these proteins in plant abiotic and biotic stress responses.

TIR1/AFB proteins involved in abiotic and biotic stress in plants.

Plant species Name Subfamily stress reference
Arabidopsis (Arabidopsis thaliana) AtTIR1 TIR1/AFB1 salt (Chen et al., 2015)
AtAFB2 AFB2/3 Salt (Iglesias et al., 2010)
AtAFB3 AFB2/3 Salt (Iglesias et al., 2010)
AtTIR1 TIR1/AFB1 Temperature (Wang et al., 2016)
AtTIR1 TIR1/AFB1 Drought (Chen et al., 2012)
AtAFB2 AFB2/3 Drought (Benny et al., 2019)
AtTIR1 TIR1/AFB1 Low Pi (Perez-Torres et al., 2008; Mayzlish-Gat et al., 2012)
AtAFB3 AFB2/3 Nitrate (Vidal et al., 2010; Vidal et al., 2013)
AtTIR1 TIR1/AFB1 Herbicide (Sheedy et al., 2006; Walsh et al., 2006; Gleason et al., 2011)
AtAFB4 AFB4/5 Herbicide (Gleason et al., 2011)
AtAFB5 AFB4/5 Herbicide (Gleason et al., 2011; Xu et al., 2022)
AtTIR1 TIR1/AFB1 Aluminum (Ruiz-Herrera and Lopez-Bucio, 2013)
AtAFB2 AFB2/3 Aluminum (Ruiz-Herrera and Lopez-Bucio, 2013)
AtAFB3 AFB2/3 Aluminum (Ruiz-Herrera and Lopez-Bucio, 2013)
At TIR1 TIR1/AFB1 Boron deficiency (Camacho-Cristobal et al., 2015; Lu et al., 2015)
AtTIR1/AFB1/AFB4/AFB5 TIR1/AFB1, AFB4/5 bacterium (Djami-Tchatchou et al., 2020)
AtAFB1 TIR1/AFB1 Fungi (Fousia et al., 2018)
AtAFB3 AFB2/3 Fungi (Fousia et al., 2018)
AtTIR1 TIR1/AFB1 Fungi (Ortiz-Castro et al., 2011)
AtTIR1/AFB2/AFB3 TIR1/AFB Fungi (Ortiz-Castro et al., 2011)
Rice (Oryza sativa) OsTIR1 TIR1/AFB1 Salt (Xia et al., 2012)
OsAFB2 AFB2/3 Salt (Xia et al., 2012)
OsAFB2 AFB2/3 Drought (Xia et al., 2012; Sharma et al., 2018)
OsTIR1 TIR1/AFB1 Drought (Xia et al., 2012; Sharma et al., 2018)
OsTIR1 TIR1/AFB1 Temperature (Sharma et al., 2018)
OsAFB2 AFB2/3 Temperature (Sharma et al., 2018)
OsAFB6 AFB6 Temperature (He et al., 2018)
OsTIR1 TIR1/AFB1 Herbicide (Guo et al., 2021)
OsAFB2 AFB2/3 Herbicide (Guo et al., 2021)
OsAFB3 AFB2/3 Herbicide (Guo et al., 2021)
OsAFB4 AFB4/5 Herbicide (Guo et al., 2021)
OsAFB5 AFB4/5 Herbicide (Guo et al., 2021)
OsTIR1 TIR1/AFB1 Virus (Jin et al., 2016)
Wheat (Triticum aestivum) TaAFB2 AFB2/3 Drought (Dalal et al., 2018)
TaTIR1 TIR1/AFB1 Temperature (Liu et al., 2022)
TaTIR1 TIR1/AFB1 Herbicide (Yu et al., 2021)
TaTIR1 TIR1/AFB1 Fungi (Su et al., 2021)
TaTIR1 TIR1/AFB1 Fungi (Gidhi et al., 2022)
Maize (Zea mays) ZmAFB2 AFB2/3 Salt (Yang et al., 2013)
ZmTIR1 TIR1/AFB1 Drought (Benny et al., 2019)
ZmTIR-like TIR1/AFB1 Fungi (Luo et al., 2014)
Soybean (Glycine max L.) GmTIR1 TIR1/AFB1 Fungi (Cai et al., 2017)
GmAFB3 AFB2/3 Fungi (Cai et al., 2017)
Melon (Cucumis melo L.) CmTIR1 TIR1/AFB1 Aphid (Sattar et al., 2016)
CmAFB2 AFB2/3 Aphid (Sattar et al., 2016)
Cucumber (Cucumis sativus L.) CSTIR1 TIR1/AFB1 Salt (Chen et al., 2017)
CsAFB2 AFB2/3 Salt (Chen et al., 2017)
Tomato (Solanum lycopersicum) SlTIR1 TIR1/AFB1 Drought (Benny et al., 2019)
SlTIR1 TIR1/AFB1 Nematode (Seah et al., 2007; Seifi et al., 2011)
Potato (Solanum tuberosum) StTIR1 TIR1/AFB1 Drought (Benny et al., 2019)
Mustard (Brassica juncea var. tumida) BjuTIR1 TIR1/AFB1 Salt (Cai et al., 2019)
BjuAFB3 Salt (Cai et al., 2019)
BjuTIR1 TIR1/AFB1 Fungi (Cai et al., 2019)
Crowtoe (Lotus corniculatus L.) LjAFB6 AFB6 nitrate (Rogato et al., 2021)
Creeping bentgrass (Agrostis stolonifera L.) AsTIR1 TIR1/AFB1 Salt (Zhao et al., 2019)
AsAFB2 AFB2/3 Salt (Zhao et al., 2019)
Fraxinus tomentosa (Fraxinus velutina Torr.) FvTIR1 TIR1/AFB1 Salt (Liu et al., 2022)

TIR1/AFB regulatory network in response to abiotic and biotic stress. TIR1/AFBs response to abiotic or biotic factors in different signal transduction pathways.

TIR1/AFB proteins are known regulators of numerous stress-related genes. The most well-studied examples of gene regulation by TIR1/AFB proteins are the Aux/IAA genes. Expression of many Aux/IAA genes in response to abiotic and biotic stress is both directly and indirectly controlled by TIR1/AFB proteins. Expression of NAC4 is also regulated by TIR1/AFB proteins in response to nitrate uptake.

The general mechanism by which TIR1/AFB proteins enhance abiotic stress tolerance is by reducing ABA accumulation, increasing the abundance of ROS scavengers, and affecting the activity of other factors such as Pi transporters. In response to biotic stress, TIR1/AFB proteins promote the expression of SA biosynthesis genes, PR genes, and PDF genes. However, more studies need to be performed to determine the role of specific TIR1/AFB members in the signaling and metabolic pathways that modulate disease resistance. As the studies highlighted in this review demonstrate, much knowledge about the role of TIR1/AFB proteins in abiotic and biotic stress responses has been generated. The next challenge for the field will be deciphering the upstream and downstream events to draw a more complete picture of TIR1/AFB-mediated regulation of plant abiotic and biotic stress responses.

 Author contributions

XC and NL concepted the topic of this manuscript and revised the manuscript, WD drafted this manuscript with YL. QL, SL and SS revised the manuscript. All authors contributed to the article and approved the submitted version.

 Funding

This work was supported by the National Natural Science Foundation of China (grant no. 32172567), Vegetable Innovation Team Project of Hebei Modern Agricultural Industrial Technology System (grant no. HBCT2018030203), Key Research & Development Project of Hebei Province (grant no. 21326309D), The Innovation Ability Training Project for Graduate Student of Hebei Province (grant no. CXZZBS2018114), and the grant from ‘Giant Plan’ of Hebei Province.

 Acknowledgments

We thank A&L Scientific Editing (www.alpublish.com) for its linguistic assistance during the preparation of this manuscript. We also thank Ma Wei, Lisong Ma, Lijun Song and Shiyao You in the preparation of the pictures in this manuscript.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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