The emerging role of biomolecular condensates in plant immunity

Abstract

Biomolecular condensates are dynamic nonmembranous structures that seclude and concentrate molecules involved in related biochemical and molecular processes. Recent studies have revealed that a surprisingly large number of fundamentally important cellular processes are driven and regulated by this potentially ancient biophysical principle. Here, we summarize critical findings and new insights from condensate studies that are related to plant immunity. We discuss the role of stress granules and newly identified biomolecular condensates in coordinating plant immune responses and plant–microbe interactions.

The authors discuss the formation and composition of several types of biomolecular condensates and their roles in regulating plant immunity.

Introduction

In cells, locally concentrated macromolecules (proteins and nucleic acids) may assemble into biomolecular condensates without a surrounding membrane. Functions and composition of such condensates are versatile, and their formation, in many cases, is dependent on liquid–liquid phase separation (LLPS) and is promoted by multivalent molecules, such as proteins containing multiple interaction interfaces or intrinsically disordered regions (IDRs) that can facilitate inter- or intramolecular interactions with multiple partners. As a membrane-independent principle of cellular organization, biomolecular condensation has recently emerged as a critical regulatory mechanism for cellular signaling in a broad range of physiological and pathological contexts in mammalian and fungal systems, and growing evidence has begun to reveal its importance in plants as well (Emenecker et al., 2020, 2021). Here, we discuss the emerging role of biomolecular condensates in regulating plant immunity.

Stress granules

Stress granules (SGs) are inducible and reversible biomolecular condensates that lack a membrane and contain translationally stalled mRNAs, translation initiation factors, and RNA binding proteins (RBPs), as well as other proteins lacking RNA binding ability (Jain et al., 2016). In humans (Homo sapiens), SGs are assembled via LLPS upon induction by a variety of endogenous and exogenous stresses, such as heat shock, oxidative stress, pathogen infection, and DNA damage (Molliex et al., 2015; Wheeler et al., 2016; Guillén-Boixet et al., 2020; Sanders et al., 2020; Yang et al., 2020). Several RBPs have been identified as integral components of plant SGs, including T-cell intracellular antigen-1/TIA-1-related protein-like RBP45/47 and UBP1 (OLIGOURIDYLATE BINDING PROTEIN1) family members, G3BP (Ras-Gap-SH3 domain binding protein)-like NUCLEAR TRANSPORT FACTOR2 family members, poly(A)-binding proteins, Tudor staphylococcal nucleases, and tandem CCCH zinc finger family members (Weber et al., 2008; Pomeranz et al., 2010; Lin et al., 2011; Bogamuwa and Jang, 2013, 2016; Sorenson and Bailey-Serres, 2014; Yan et al., 2014; Gutierrez-Beltran et al., 2015, 2021; Nguyen et al., 2016; Krapp et al., 2017; Nguyen et al., 2017). Using these RBPs as the proxy for plant SGs, studies have begun to reveal potential interactions between biotic stresses and plant SGs.

SG formation is frequently observed during viral infection because SGs are intimately linked with the cellular translation capacity that is required for viral replication (reviewed by Protter and Parker, 2016). Therefore, proper regulation of SG formation is critical for defense against viral infection, although the detailed mechanism in plants remains largely unclear. It was reported by Hafren et al. (2015) that Nicotiana plumbaginifolia UBP1 could be recruited into the Potato virus A (PVA)-induced granules when transiently expressed in Nicotiana benthamiana. Interestingly, although UBP1 suppresses PVA translation, it is also required for a high level of PVA infection both locally and systemically. This complicated effect is probably due to a positive contribution of UBP1 to the formation of PVA-induced granules, which also play a role in interfering with the host silencing machinery (Hafren et al., 2015). Plant viruses have also developed effective strategies to disturb host SG formation. For example, heat stress-induced TZF1 granule formation in N. benthamiana can be compromised by the viral nonstructural protein 3 (nsP3_31) from the Semliki Forest virus. TZF1 appears to be recruited by nsP3_31 out of SGs into a network-like structure (Krapp et al., 2017). Similarly, in N. benthamiana, the SG component UBP1B restricts Potato virus X infection; however, this virus is able to suppress SG formation through relocating UBP1B (Robles-Luna et al., 2020).

Aside from viruses, microbe-associated molecular patterns (MAMPs) are an important type of plant biotic stress signal. In Arabidopsis (Arabidopsis thaliana), activation of plant pattern-triggered immunity (PTI) by elf18, an elicitor of PTI derived from bacterial elongation factor-Tu, enhances the translational efficiency of a specific set of genes containing the R motif, a consensus 5′-sequence that almost exclusively comprised purines. The plant SG marker protein PAB2 (Sorenson and Bailey-Serres, 2014) directly binds the R motif and positively regulates elf18-induced resistance against the bacterial pathogen Pseudomonas syringae (Xu et al., 2017). However, it remains to be determined whether elf18 promotes the formation of PAB2-positive SGs and whether the translational regulation of R motif-containing transcripts requires the formation of such condensates. Recently, Tabassum et al. reported that activation of PTI in Arabidopsis by flg22, another MAMP derived from bacterial flagellin, leads to MITOGEN-ACTIVATED PROTEIN KINASE 3/6 (MPK3/6)-dependent phosphorylation of TZF9 and reduces its stability. They also found that TZF9 directly interacts with PAB2 and promotes its relocation to SGs. Therefore, MPK3/6 may control defense gene translation through modulation of TZF9 stability and subsequently influence the assembly of TZF9-PAB2-positive SGs (Tabassum et al., 2020).

Besides the participation in the immune responses elicited by viruses and MAMPs, plant SGs have also been implicated in the regulation of autoimmunity. In Arabidopsis, RH6/8/12, DExD/H-box helicase 1/DEAD-box helicase 6-like RNA helicases, dynamically associate with hypoxia-induced SGs and regulate SG formation. RH6/8/12 repress the translation of defense-related genes and promote the translation of growth-related genes. Consistently, the Arabidopsis rh6 8 12 triple mutant displays a typical autoimmune phenotype and enhanced resistance to the virulent oomycete pathogen Hyaloperonospora arabidopsidis (Chantarachot et al., 2020). Therefore, the study by Chantarachot et al. suggests an autoimmune-related function of SGs induced by abiotic stress conditions. In line with this idea, a recent profiling of heat stress-induced SG proteome in Arabidopsis also revealed several defense-related components (Kosmacz et al., 2019).

As the role of plant SGs in biotic stress responses begins to be elucidated, several fundamental questions remain. First, although cumulating studies have supported LLPS as a key force driving the assembly of SGs in yeast (Saccharomyces cerevisiae) and human cells, more direct evidence is needed to support a similar mechanism in plants. Second, systematic studies are needed to determine whether different types of biotic stresses, including challenges from microbes, pests, and parasitic plants, can trigger the formation of SGs and/or modulate their dynamics. Also, the roles of phytohormones such as salicylic acid (SA), methyl jasmonate, and ethylene during this process need to be clarified. Third, the protein and RNA composition of immune-related plant SGs need to be profiled for a better understanding of the function of plant SGs in defense activation. Finally, we also need to understand what external or internal signals trigger SG assembly upon biotic stresses and how SGs are disassembled when the stress condition is relieved.

SA-induced NPR1 condensates

NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a master regulator of SA-mediated plant responses, including defense activation and cell survival promotion (Wang et al., 2006; Fu et al., 2012). It was recently reported that high levels of SA promote the formation of cytoplasmic SA-induced NPR1 condensates (SINCs) in plant cells (Zavaliev et al., 2020). In SINCs, NPR1 recruits CUL3 E3 ligase to potentially ubiquitinate and mediate degradation of SINC-localized cell death-promoting regulators, including NLR receptors, EDS1, and WRKY54/70 transcription factors, and thus promote cell survival. This NPR1 function appears to be distinct from the nuclear NPR1, which functions as a transcription coactivator for defense gene activation. In addition to high levels of SA, SINCs also form in response to a range of stresses, including heat, oxidative, and DNA damage stresses, suggesting a diverse role of SINCs in plant stress responses (Zavaliev et al., 2020).

NPR1 contains multiple cysteine residues and forms intermolecular disulfate bonds that regulate the protein’s subcellular localization and activity (Mou et al., 2003). SINC formation requires the cysteine-dependent and redox-sensitive IDR domain localized in the C-terminus of NPR1 protein (Zavaliev et al., 2020). A recent study in tomato (Solanum lycopersicum) found that shoot apical meristem-produced H₂O₂ promotes the formation of disulfide bonds and IDR-dependent phase separation of the TERMINATING FLOWER (TMF) transcription factor. The resulting TMF-containing condensates sequester the promoter of floral identity gene ANANTHA and prevent floral transition (Huang et al., 2021b). Both SA and ROS are produced during infection by biotrophic pathogens and play a critical role in regulating cellular redox and immune signaling. We propose that redox-driven condensation of immune molecules plays a more important role in modulating their activities than previously appreciated.

Posttranslational modifications (PTMs) have been shown to play an important role in modulating the dynamics of biomolecular condensates. In animals, phosphorylation and ubiquitination of core SG proteins are common strategies to tune the formation of SG by altering specific protein–protein interactions (Dao et al., 2018; Wang et al., 2019; Yang et al., 2020). SA-induced dephosphorylation at two serine residues on NPR1 (S55/59) is a prerequisite for both NPR1 nuclear translocation and SINC formation in the cytoplasm. Subsequent SUMOylation of NPR1 may contribute to NPR1 condensate formation in the nucleus but negatively regulate SINC formation (Saleh et al., 2015; Zavaliev et al., 2020). Plants exploit versatile PTMs on immune regulators to achieve rapid and precise immune signaling (Withers and Dong, 2017). Future research will establish critical roles of various types of PTMs in regulating the formation of biomolecular condensates during plant immune signaling.

GBPL defense-activated condensates

Besides SINCs, another type of immune-related condensates was recently defined in Arabidopsis. The Arabidopsis guanylate-binding protein (GBP)-like GTPase (GBPL), GBPL3, was found necessary for resistance against Pseudomonas syringae, and the overexpression of GBPL3 confers autoimmunity (Huang et al., 2021a). Interestingly, different from human GBPs, about half of plant GBPL proteins (including GBPL3) evolved an additional IDR domain in their carboxyl terminus, and this feature can be traced back to unicellular green algae. Indeed, both exogenous SA application and pathogen infection increase the nuclear distribution of GBPL3 and promote rapid formation of GBPL3-positive nuclear condensates, which are defined as GBPL defense-activated condensates (GDACs). Both the IDRs and the GTPase activity were found required for GBPL3-mediated resistance and LLPS-dependent GDAC formation. The nuclear distributed GBPL3 was shown to associate with promoters of defense genes and interact with members of the Mediator complex and RNA polymerase II. It was proposed that phase-separated GBPL3 helps consolidate RNAPII-Mediator interaction and thus promote defense gene expression (Honkala et al., 2019; Huang et al., 2021a). Mammalian GBP proteins are involved in diverse cellular processes, including endomembrane trafficking, autophagy, immune activation, intracellular pathogen restriction, cytoskeleton dynamics, cell death, and cell cycle progression (Ngo and Man, 2017; Honkala et al., 2019; Huang et al., 2019). It will be interesting to further investigate the direct signal that promotes GDAC formation and to determine whether GBPL3 and GDACs play a role in regulating other cellular processes in plants.

Nuclear transport receptors

Nuclear transport receptors (NTRs), particularly importin-βs (IMBs), are functionally conserved cargo carriers that mediate bidirectional nucleocytoplasmic transport of macromolecules in eukaryotes (Tamura and Hara-Nishimura, 2014). IMBs are capable of interacting with phenylalanine–glycine (FG) repeats-containing nucleoporins that compose the selective barrier of the nuclear pore complex. FG Nups are intrinsically disordered and multivalent and can potentially form a gel-like phase within the nuclear pore complex central channel, with which IMBs can specifically interact and pass through (Schmidt and Gorlich, 2016). Recent advances in animal research found that IMB2, which mediates nuclear transport of cargo bearing the proline–tyrosine (PY)-NLS signal, can prevent aberrant phase transition of a myriad of IDR-containing RBPs. For example, in animals, IMB2 masks the low-complexity domain of RBP FUS, weakening the interaction of FUS with SGs to prevent the formation of disease-associated cytoplasmic condensates. This chaperoning function is independent of the nuclear transport activity of IMB2, and it can lead to a complete disaggregation of SGs, thus reducing cytotoxicity and mitigating neurodegenerative diseases (Guo et al., 2018, 2019; Hofweber et al., 2018; Qamar et al., 2018; Yoshizawa et al., 2018). This dual transport and chaperoning activity of NTRs suggests their unique property and power in engaging and regulating biomolecular condensates in cells. The Arabidopsis IMB2 homolog was also reported to transport RBPs (Ziemienowicz et al., 2003). Arabidopsis imb2 mutant plants show stunted growth that mimics an autoimmune phenotype (Cui et al., 2016), suggesting a potential role in regulating immune and stress responses. Other NTRs have been recently reported to be intimately involved in regulating immune strength and the activity of critical immune factors in plants (Li and Gu, 2020; Ludke et al., 2020; Jia et al., 2021; Xu et al., 2021). Future studies will certainly shed light on whether NTRs directly participate in regulating stress-related biomolecular condensates in plant cells and how they could be engineered to balance immunity and crop growth/yield through fine tuning immune strength.

Pathogen effectors

On the pathogen side, a virulent strategy was recently found to rely on LLPS-based biomolecular condensation. Many phytopathogens can secrete effector proteins into plant cells to hijack host immune systems. Intriguingly, predicted IDRs are highly enriched in effectors secreted by phytopathogens and were previously thought to facilitate their secretion and interactions with host proteins by providing structural flexibility (Marin et al., 2013). However, a recent study revealed the functional importance of the effector IDR in promoting virulence. XopR, an IDR-containing type-III effector protein from Xanthomonas campestris, subverts Arabidopsis actin cytoskeleton dynamics beneath the plasma membrane by interacting with actin and multiple actin-binding proteins and promoting host actin nucleation through LLPS (Sun et al., 2021). Thus, LLPS appears to be well exploited by both pathogens and hosts in their battle.

Growing evidence supports the idea that formation of biomolecular condensates (via LLPS) is a critical biophysical mechanism that regulates a broad spectrum of cellular processes and signaling events beyond immune activation in plants (Fang et al., 2019; Powers et al., 2019; Jung et al., 2020; OuYang et al., 2020; Huang et al., 2021b). Identification of more stress-related biomolecular condensates in plants and the elucidation of mechanisms controlling their formation, dynamics and regulation by internal and external signals represent an exciting new research area in plant science in the future.

 

The manuscript was writtern by W.W. and Y.G.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) are: Wei Wang (oneway1985@pku.edu.cn) and Yangnan Gu (guyangnan@berkeley.edu).