3.1. Signaling Pathways Controlling Pathogen-Induced Callose Deposition

As part of PTI, PAMP-induced callose deposition is under the control of plant PRRs. Activity of the downstream pathways is marked by common signaling events, such as anion fluxes, protein phosphorylation cascades, accumulation of ROS, and defense gene induction. In Arabidopsis, ROS act as positive signals in Flg22-induced callose [31,32] (Figure 1A). The RNAi regulatory protein Argonaute1 also generates various miRNA signals that stimulate or repress Flg22-induced callose [33] (Figure 1A). Flg22-induced callose in Arabidopsis also requires intact biosynthesis of 4-methoxylated indole glucosinolates [34] (Figure 1A), suggesting that these secondary metabolites or break-down products play a crucial role in the regulation of callose. Interestingly, Flg22- and chitosan-induced callose differ in the requirement for the NADPH oxidase RBOHD, the glucosinolate regulatory enzymes PEN2, and the callose synthase PMR4 [35]. The rbohD mutant accumulated reduced levels of Flg22-induced H₂O₂ and failed to deposit enhanced levels of callose upon treatment with Flg22. However, the rbohD mutant deposited normal levels of callose in response to chitosan, even though chitosan-induced H₂O₂ was reduced. Likewise, the pen2-2 mutant failed to deposit enhanced callose in response to Flg22, whereas chitosan induced statistically significant enhancements in callose deposition. The pmr4-1 mutant deposited dramatically reduced levels of basal callose and failed to respond to Flg22. However, chitosan still elicited a residual callose response in pmr4-1 plants, indicating that one or more other callose synthases than PMR4 are responsible for a portion of chitosan-induced callose. These results demonstrated that PAMP-induced callose is controlled by distinct signaling pathways.

SA plays an important role in pathogen-induced plasmodesmata closure and associated callose deposition in Arabidopsis [36]. Direct exogenous application of SA or bacterial infection induce plasmodesmata closure and callose deposition. SA pathway mutants are impaired in this response. The SA- or pathogen-induced plasmodesmata closure and associated callose deposition requires an ENHANCED DESEASE RESISTANCE1(EDS1)– and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1)–dependent SA pathway and is dependent on the regulator of plasmodesmal gating PLASMODESMATA-LOCATED PROTEIN5(PDL5) [36](Figure 1B). These results indicate that SA is a crucial signal in the regulation of cell-to-cell communication and trafficking via plasmodesmata in plant cells during innate immune responses.

Abscisic acid (ABA) signaling has an important effect on induced or primed callose deposition. Callose priming induced by the nonprotein amino acid β-amino butyric acid (BABA) requires an intact ABA-dependent pathway in Arabidopsis. BABA-induced resistance in Arabidopsis against Plectosphaerella cucumerina is known to be mediated by callose priming. Indole-3-carboxylic acid (ICOOH, also known as I3CA) mediates BABA-induced resistance in Arabidopsis against P. cucumerina. I3CA treatment increased ABA levels, which activates a starch amylase (BAM1) to trigger augmented callose deposition against P. cucumerina [37]. A similar role of ABA and starch metabolism has also been reported in mycorrhiza-induced callose priming in tomato plants upon infection by necrotrophic fungal pathogen B. cinerea [24]. Intriguingly, ABA can enhance or repress Flg22-induced callose deposition in Arabidopsis depending on growth conditions [35] (Figure 1A). Under most growth conditions, ABA has a stimulatory effect on Flg22-induced callose deposition. However, ABA reduced basal and PAMP-induced callose at low light intensity, high sucrose concentration and vitamins (Figure 1A). ABA also represses Flg22-induced callose in hydroponic Arabidopsis grown with the a relatively high concentration of sucrose in the growth medium (0.5%) [34,35] (Figure 1A). These results suggest that ABA’s role in defense-related callose deposition and priming is influenced by carbohydrate metabolism.

3.2. Biogenesis and Activation of Pathogen-Responsive Callose Synthases

As a large transmembrane protein, GSL5/PMR4 belongs to the family of glucan synthases that include plant cellulose synthases. Plant cellulose synthases are synthesized on the endoplasmic reticulum (ER) and go through the ER quality control system for proper folding and modification before being assembled into complexes in the Golgi apparatus and transported to the plasma membrane through the Golgi- and post-Golgi trafficking system [38]. Many proteins involved in plant immune responses, including pattern-recognition receptors and extracellular defense-related proteins, are also synthesized on the ER, go through the ER quality control, and ultimately are transported to their destinations through the secretory pathway. We have recently shown that two Arabidopsis homologs of UBAC2, a conserved ER protein implicated in ER quality control and ER-associated degradation (ERAD), play a critical role in pathogen- and PAMP-induced callose deposition [39]. The UBAC2 proteins interact with the plant-specific PAMP-INDUCED COILED COIL (PICC) protein, which is also localized in the ER and is important for pathogen- and PAMP-induced callose deposition (Figure 2). Therefore, the evolutionarily conserved UBAC2 and plant-specific PICC proteins are critical components of an ER pathway with an important role in plant immunity by regulating pathogen-induced callose deposition. The compromised phenotypes of the ubac2 and picc mutants in PTI and pathogen/PAMP-induced callose deposition could be rescued by overexpression of PMR4, suggesting that disruption of UBAC2 and PICC genes may reduce the accumulation of PMR4 callose synthase in the ubac2 and picc mutants, leading to defects in pathogen/PAMP-induced callose deposition [39]. Further analysis using both GFP- and myc-labeled PMR4 indeed discovered that disruption of UBAC2 or PICC reduced the levels of plasma-membrane-localized PMR4 [39]. These results indicate that the evolutionarily conserved UBAC2 proteins function in coordination with a plant-specific protein PICC in the positive regulation of the biogenesis of PMR4, either by stabilizing the callose synthase or degrading a negative regulator of PMR4 stability (Figure 2).

Figure 2. Synthesis, transport, activation, and recruitment of Arabidopsis PMR4 callose synthase during plant defense responses. Some of the important factors identified to be involved in these processes are indicated.

In addition, there is evidence for PMR4 activation in response to pathogen infection or other stress signals. In uninfected PMR4-overexpression lines, no significant increase in the callose synthase activity or callose deposition was observed [40]. In yeast, activation and translocation of callose synthases involve GTPases [41,42]. In Arabidopsis, PMR4 interacts with GTPase RabA4c [43] (Figure 2). Similar to PMR4 overexpression, RabA4c overexpression leads to complete penetration resistance to the virulent powdery mildew fungal pathogen in a PMR4-dependent manner [43]. By contrast, overexpression of a dominant negative form of RabA4c fails to increase callose deposition or penetration resistance [43]. These results indicate that the RabA4c GTPase plays a critical role in the activation and translocation of PMR4 during pathogen-induced callose deposition. As a transmembrane protein synthesized on the ER, PMR4 is likely subjected to additional regulatory mechanisms for its biogenesis, trafficking, and activation to ensure biosynthesis of callose in a timely manner.

3.3. Transport Processes in Pathogen-Induced Callose Deposition

Exocytosis is a process by which cells are able to not only move materials from within a cell to the exterior of the cell, but also insert membrane proteins (such as ion channels and cell surface receptors), lipids, and other components into the cell membrane [44]. Vesicles containing these membrane components fully fuse with and become part of the plasma membrane. The exocyst, an octameric protein complex involved in exocytosis, tethers and spatially target post-Golgi vesicles to the plasma membrane prior to vesicle fusion [44]. It has been demonstrated that the exocyst subunit EXO70H4 is important in the deposition of the callose-rich cell wall layer for biogenesis of the trichome secondary cell wall [45,46]. PMR4 is a callose synthase responsible for the synthesis of callose in the trichome and PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70h4-1 mutant. Significantly, EXO70H4 expression is induced by pathogen elicitor Flg22, which induces callose deposition, in epidermal pavement cells [46]. These results raise a strong possibility that EXO70H4-dependent exocytosis is also involved in the trafficking of newly synthesized PMR4 callose synthase proteins from the Golgi apparatus to the plasma membrane (Figure 2).

Another transport process important for defense-related callose deposition is the recruitment of callose synthases protein such as PMR4 in Arabidopsis from the plasma membrane and transport in vesicle-like bodies to the site of attempted penetration after fungal infection (Figure 1). There is strong evidence that multivesicular bodies (MVBs) are involved in the transportation and delivery of defense components to the forming papilla. Studies have showed accumulation of MVBs and cell wall-associated paramural bodies (PMBs) in the vicinity of pathogen-induced papillae [47,48,49]. PMBs, which are situated between the cell wall and the plasma membrane, are likely to have resulted from the fusion of MVBs with the plasma membrane [50]. Plant MVBs and PMBs have been observed near papillae in plant cells infected by pathogenic fungal, bacteria and nematodes for delivery of defense-related molecules including phytoalexins, callose and ROS to papillae [47,48,51,52,53]. In addition, in plant interaction with filamentous pathogens, each haustorium formed upon successful penetration is surrounded by the plasma membrane of the plant cell termed extrahaustorial membrane (EHM), which is likely synthesized de novo [54]. In tobacco (Nicotiana benthamiana) cells invaded by oomycete pathogen P. infestans, the Rab7 GTPase RabG3c MVB marker protein, but not a tonoplast-localized sucrose transporter, is recruited to the EHM [55]. In Arabidopsis, the Rab5 GTPase, also an MVB marker, accumulates in the EHM after infection with a powdery mildew fungus [56] (Figure 2). Thus, specific rerouting of MVBs from the vacuole to the host-pathogen interface may participate in the formation or modulation of the EHM. In barley, MVBs contained the ADP-ribosylation factor (ARF) GTPase ARFA1b/1c that was important for callose deposition at powdery mildew penetration sites [51]. RNAi knockdown or expression of a dominant negative ARFA1b/1c variant abolished callose accumulation at penetration sites and resulted in increased fungal penetration success. ARFA1b/1c localized to an endosomal MVB compartment that accumulated at fungal penetration sites prior to the accumulation of callose. Interestingly, the Arabidopsis ARF-GEF (guanine nucleotide exchange factor) MIN7 is required for normal levels of callose deposition in response to the Pseudomonas syringae pv. tomato ΔCEL mutant, suggesting that an ARF-dependent vesicle trafficking process may also play a role in callose deposition at sites of pathogen detection in Arabidopsis [57] (Figure 2).