Fungal Effector Protein Functions: History
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Contributor:
María Victoria Aparicio Chacón
,
Judith Van Dingenen
,
Sofie Goormachtig

Plants are colonized by various fungi with both pathogenic and beneficial lifestyles. One type of colonization strategy is through the secretion of effector proteins that alter the plant’s physiology to accommodate the fungus. The oldest plant symbionts, the arbuscular mycorrhizal fungi (AMF), may exploit effectors to their benefit. Genome analysis coupled with transcriptomic studies in different AMFs has intensified research on the effector function, evolution, and diversification of AMF. However, of the 338 predicted effector proteins from the AM fungus Rhizophagus irregularis, only five have been characterized, of which merely two have been studied in detail to understand which plant proteins they associate with to affect the host physiology. 

  • secretome
  • effector proteins
  • arbuscular mycorrhizal fungi

1. Introduction

Approximately 80% of land plants associate with AMF [1][2]. Throughout this intimate root association, a complex transcriptional and physiological reprogramming of the plant is established to ensure the formation of arbuscules within the cortical cells that act as the functional unit of the symbiosis [3]. In these arbusculated cells, extensive trafficking of molecules takes place, with host plants delivering up to 20% of their fixed carbon to the symbiont in exchange for inorganic phosphate, nitrogen, water, and micronutrients [4][5][6][7].
Despite being the oldest plant symbiosis in the world [8], little is known about the fungal molecules that fine-tune this beneficial relationship in parallel to or downstream of the host perception of chitin oligosaccharides and lipochitooligosaccharides [9], also called Myc factors, an event governing fungal colonization [3]. One way microorganisms communicate with host cells is through the secretion and translocation of effector proteins, which have been intensively studied in plant-pathogen interactions [10][11][12] and are expected to modulate important aspects of the AM symbiosis as well [13][14]. These effector proteins have been shown to act in the plant cell apoplast or intracellularly, where they often interact with diverse host biomolecules to suppress immunity and allow fungal accommodation [10][15][16].
Several strategies, combining both in silico prediction and wet-lab experiments, have been proposed to highlight the effectors of interest for a particular plant-microbe interaction [17]. In the last decade, genomic and transcriptomic studies in different AMF strains, such as Rhizophagus irregularis [18][19][20][21][22], Gigaspora rosea [23][24], and R. clarus [21][25] have been published, underlining the existence of hundreds of potential effector genes, some of which are predicted to be conserved among different AMF strains [21][23]. Additionally, transcriptome analysis of different hosts and tissues colonized by R. irregularis shed light on the common and host-specific effectors used by this mycorrhizal fungus [26]. From these hundreds of potential effectors, a total of five R. irregularis effector proteins have been in-depth characterized for their role in AM symbiosis in the model legume Medicago truncatula [27][28][29][30][31]. Thus, hundreds of potential AMF effector proteins remain to be characterized that may play a relevant role in modulating the association.

2. Fungal Effector Protein Functions: Suppression of Defense and Niche Occupation

In general, pathogenic and beneficial plant-colonizing organisms utilize effector proteins to suppress immunity and to allow niche establishment [32][33]. To successfully colonize their plant hosts, microbes, including beneficial ones, must overcome the two major layers of plant immunity, which include membrane-localized and intracellular surveillance systems [17]. Cell surface perception of molecules derived from microbes or damaged plant cells via host pattern recognition receptors (PRRs) activates the pattern-triggered immunity (PTI) and downstream defense responses [34][35]. Depending on the nature of these molecules, they can be classified as microbe-associated molecular patterns (MAMPs), including chitin-containing molecules derived from the fungal wall, or as plant damage-associated molecular patterns (DAMPs) [36][37]. When microbes successfully overcome the first plant defense barrier, a second layer of intracellular immunity is initiated [38][39].
Microbes have developed multiple strategies to interfere with or avoid PTI recognition, including the secretion of specialized effector proteins [38]. Proteinaceous effectors are small secreted proteins (SSPs) that can regulate various aspects of the host physiology through selective binding to host plant macromolecules, such as proteins, DNA, and RNA [40]. A large collection of effectors involved in plant host immunity avoidance or suppression has been described for a variety of fungal and oomycete plant pathogens, such as the R×LR effector family or the Crinkler (CRN) effector in the pathogenic oomycete Phytophthora infestans [33][41][42][43][44]. In addition, effector proteins manipulate plant cells to establish the growth niche, and some change the nutrient status of the plant in favor of the pathogen, for instance by increasing the sugar efflux to boost microbial growth [45]. One of the best-known examples is the bacterial pathogen Xanthomonas oryzae, which infects Oryza sativa (rice) and secretes the PthXo1 effector into plant cells, where it activates the transcription of SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 11 (OsSWEET11), which encodes a protein that exports sugars to the apoplast to feed the pathogen [45][46]. Although the direct association of fungal and oomycetes effector proteins to SWEET elements is lacking, the involvement of SWEET genes in fungal susceptibility has been recently elucidated [47][48][49]. For instance, the Arabidopsis thaliana Atsweet14 mutants display low susceptibility to the infection by the necrotrophic fungus Botrytis cinerea, suggesting that this transporter is involved in feeding the fungus to support its development [47].
Based on structural analyses and functional validations, a wide range of in silico tools are now available to effectively predict fungal effector protein features. Key effector characteristics include (i) the presence of an N-terminal signal peptide of 18–30 amino acids that guides their conventional secretion outside the fungus [41]; (ii) the generally small size of less than 300 amino acids [50]; and (iii) the absence of a transmembrane domain to guarantee their presence in the extracellular space or inside the host cell [17][51]. To be directed to specific plant subcellular compartments, intracellular effectors often display specific amino acid sequences, such as nuclear localization signals (NLSs) or mitochondrial and chloroplast transient peptides [21][52]. Moreover, fungal effector proteins are frequently hallmarked by the presence of intrinsic disorder regions (IDRs) and the absence of characterized functional protein domains [53]. This lack of similarity to functionally characterized protein domains further challenges the identification of fungal effectors, because little information about their function can be extrapolated from the sequence [54].
Subsequently, the elucidation and characterization of the plant target are essential to explore the molecular processes in which effector proteins are involved. Indeed, studies of the targeted host proteins, DNA, or RNA regions of diverse fungal effectors have elucidated their mode of action in different plant hosts [42][55][56]. For example, the rust fungus Melampsora larici-populina secretes the nuclear-localized effector protein Mlp124478 that binds to the promoter region of the basic leucine zipper motif transcription factor (TF) TGA1a, boosting its transcription to suppress defense genes, such as the signaling gene JAZ1 or the defense-related TF WRKY18 in Arabidopsis [55]. Similarly, the two nuclear-localized effector proteins MoHTR1 and MoHTR2, from the rice blast pathogen Magnaporte oryzae, carry zinc finger DNA-binding domains that associate with the promoters of immunity-related genes encoding the plant TFs OsMYB4 and OsWRKY45 to repress their expression [42]. Another example of a functionally characterized effector is the arginine-rich RNA-binding effector protein Pst_A23 from the pathogenic fungus Puccinia striiformis of Triticum aestivum (wheat) [56]. Pst_A23 accumulates in nuclear speckles, where it manipulates the splicing of the leucine-rich repeat (LRR) receptor-like serine/threonine protein kinase mRNA TaXa21-H and the WRKY TF TaWRKY53 by binding to the specific motifs M1 (5-GA_GAA-3) and M2 (5-UUCUUU-3), respectively [56]. These effector protein-RNA tandem complexes decrease the levels of the alternatively spliced versions of TaXa21-H and TaWRKY53, both encoding proteins that are positively involved in plant adaption to various stresses, thereby reducing the plant defense responses [56].
Besides DNA and RNA, fungal effectors target plant proteins as well, such as the conserved necrosis-inducing secreted protein 1 (NIS1) effector from the fungal endophyte Colletotrichum tofieldiae and the rice blast fungus M. oryzae [32]. NIS1 interacts with the PRR-associated serine/threonine protein kinases BAK1/SERK3 and the Botrytis-induced kinase 1, inhibiting their kinase activities and preventing the PAMP-triggered reactive oxygen species (ROS) burst, resulting from the PTI activation in Arabidopsis, rice, and Hordeum vulgare (barley) [32].

This entry is adapted from the peer-reviewed paper 10.3390/ijms24119125

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