bState Key Laboratory of Cotton Biology, Institute of Cotton Research of the Chinese Academy of Agricultural Sciences, Anyang 455000, China
bState Key Laboratory of Cotton Biology, Institute of Cotton Research of the Chinese Academy of Agricultural Sciences, Anyang 455000, China
*
https://orcid.org/0000-0003-2975-9953

This work was sponsored by the “Seven Crop Breeding” National Major Project (grant no. 2016YFD0101006), the Genetically Modified Organism Breeding Major Project (grant no. 2018ZX08005001-002), and the State Key Laboratory of Cotton Biology Open Fund (grant no. CB2017B03).

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The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yuxia Hou (houyuxia

Y.H., F.L., and N.L. conceived and designed the experiments; N.L., Y.S., and Y.P. executed the experiments; N.L. analyzed the data; N.L. wrote the manuscript; X.Z., P.W., and X.L. contributed reagents, materials, and analysis tools.


Plant Physiology, Volume 176, Issue 3, March 2018, Pages 2202–2220, https://doi.org/10.1104/pp.17.01399
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Abstract

Pectins are major components of the primary plant cell wall, which functions as the primary barrier against pathogens. Pectin methylesterases (PMEs) catalyze the demethylesterification of the homogalacturonan domains of pectin in the plant cell wall. Their activity is regulated by PME inhibitors (PMEIs). Here, we provide evidence that the pectin methylesterase-inhibiting protein GhPMEI3 from cotton (Gossypium hirsutum) functions in plant responses to infection by the fungus Verticillium dahliae. GhPMEI3 interacts with PMEs and regulates the expression of a specific fungal polygalacturonase (VdPG1). Ectopic expression of GhPMEI3 increased pectin methyl esterification and limited fungal disease in cotton, while also modulating root elongation. Enzymatic analyses revealed that GhPMEI3 efficiently inhibited the activity of cotton GhPME2/GhPME31. Experiments using transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the GhPMEI3 gene under the control of the CaMV 35S promoter revealed that GhPMEI3 inhibits the endogenous PME activity in vitro. Moreover, the enhanced resistance to V. dahliae was associated with altered VdPG1 expression. Virus-induced silencing of GhPMEI3 resulted in increased susceptibility to V. dahliae. Further, we investigated the interaction between GhPMEI3 and GhPME2/GhPME31 using inhibition assays and molecular docking simulations. The peculiar structural features of GhPMEI3 were responsible for the formation of a 1:1 stoichiometric complex with GhPME2/GhPME31. Together, these results suggest that GhPMEI3 enhances resistance to Verticillium wilt. Moreover, GhPMEI3-GhPMEs interactions would be needed before drawing the correlation between structure-function and are crucial for plant development against the ever-evolving fungal pathogens.


Plant cell walls are highly heterogeneous extracellular structures that contain three major classes of polysaccharides—cellulose, hemicellulose, and pectin—as well as phenolic compounds and cell wall proteins (Dedeurwaerder et al., 2009; Nguyen et al., 2016). Pectin is the most complex of these polysaccharides; it is located in the primary cell wall and constitutes the principal component of the middle lamella (Johansson et al., 2002). It is composed of three fractions of galacturonans: homogalacturonan (HG), rhamnogalacturonan-I, and minor amounts of rhamnogalacturonan-II (Caffall and Mohnen, 2009). HG is a chain composed of α-1,4-linked-d-GalUA units; it is synthesized in the Golgi and then secreted to the cell wall in a highly methyl esterified (up to 80%) form (Sterling et al., 2001). There, pectin methylesterases (PMEs; EC 3.1.1.11), which are cell wall enzymes, deesterify HG, releasing methanol and protons (Dedeurwaerder et al., 2009).

In cotton (Gossypium hirsutum), PMEs are encoded by a large multigene family. Based on their structure, PMEs have been classified into two enzyme types, both of which possess a conserved PME domain (Pfam 01095; Sénéchal et al., 2015). PMEs that contain a PRO region at the N-terminal end of the catalytic domain have been designated as type I (Micheli, 2001). The PRO region shares similarity with the PME inhibitor domain (Pfam 04043; Pelloux et al., 2007) that is presumed to be cleaved from the mature catalytic portion of the protein during secretion (Dorokhov et al., 2006; Dedeurwaerder et al., 2009). The PRO domain mediates the retention of PMEs in the Golgi compartment, and regulates the enzymatic activity of PME through a posttranslational mechanism (Wolf et al., 2009; Sénéchal et al., 2015). PMEs lacking the PRO region are type II enzymes and are produced by bacteria, fungi, Physcomitrella patens, and higher plants (Dedeurwaerder et al., 2009).

Plant PMEs play an important role in preparing the substrate for processing by polygalacturonases and pectate lyases during cell wall metabolism (Johansson et al., 2002). Plant PMEs are typically multigene-encoded isoenzymes that are involved in plant defense against pathogens (Raiola et al., 2011; Bellincampi et al., 2014; Bethke et al., 2014; Lionetti et al., 2017). It is thought that plant PMEs remove methylesters in a block-wise fashion (single-chain mechanism) generating continuous, deesterified GalUA residue domains (Willats et al., 2001). However, PMEs also exist in bacteria and in fungal pathogens. There, the PME mode of action for removing methylesters is random (multiple-chain mechanism; Kohn et al., 1983; Limberg et al., 2000), except for the fungal PME from Trichoderma reesei (Markoviě and Kohn, 1984).

Moreover, the three-dimensional structures of the plant and microbial PMEs are very similar, as the proteins are all composed of right-handed β-helices. A cleft in the surface of the β-helix structure constitutes the active region of the protein. In plant PMEs, the active site cavity is relatively shallow, with no obvious steric barriers, while the active site cavity of the Erwinia chrysanthemi PME is relatively deep (Jenkins et al., 2001). Since PMEs contain neither an α/β hydrolase fold nor a catalytic Ser-His-Asp triad, they are considered a new type of hydrolases. They appear to be carboxylate hydrolases containing two Asp residues in the active site (Jenkins et al., 2001; Johansson et al., 2002).

PME inhibitors (PMEIs) belong to a large multigene-encoded protein family, PF04043, that includes invertase inhibitors (INHs). PMEIs harbor four conserved Cys residues engaged in disulfide bridge formation, and an up-and-down four-helical bundle fold, which is similar to INHs (Di Matteo et al., 2005; Lionetti et al., 2015). PME activity is efficiently regulated by endogenous PMEIs. PMEIs are targeted to the extracellular matrix and typically inhibit plant PMEs by forming a specific and stable complex with 1:1 stoichiometry (Lionetti et al., 2014). A high-resolution three-dimensional structure of a PME-PMEI complex revealed that PMEI covers the pectin-binding cleft of PME and conceals the putative catalytic sites. Thus, it prevents the substrate from approaching the cleft (Di Matteo et al., 2005; Hothorn et al., 2010).

It is thought that most of the important interacting residues are conserved in plant PMEs, but not in fungal and bacterial enzymes; hence, PMEIs are ineffective against microbial enzymes (Di Matteo et al., 2005). Nevertheless, the PMEI from pepper (Capsicum annuum) exhibits antifungal activity against Fusarium oxysporum f. sp. matthiole, Alternaria brassicicola, and Botrytis cinerea (An et al., 2008). Virus-induced gene silencing of CaPMEI1 results in enhanced susceptibility to Xanthomonas campestris pv vesicatoria (An et al., 2008). The effectiveness of PMEIs in controlling endogenous PME activity was first demonstrated in planta by overexpressing AtPMEI-1 and AtPMEI-2 in Arabidopsis. The disease symptoms caused by B. cinerea and Pectobacterium carotovorum were considerably reduced in transgenic plants (Lionetti et al., 2007). Later, kiwi (Actinidia deliciosa) PMEI was shown to limit fungal infections caused by Bipolaris sorokiniana, F. graminearum, and Claviceps purpurea in durum wheat (Triticum aestivum; Volpi et al., 2011, 2013). Moreover, PMEIs counteract the action of plant PMEs and affect the susceptibility of plants to viruses. For example, Tobacco mosaic virus symptoms are reduced, and its systemic movement is limited in tobacco (Nicotiana tabacum) that heterologously expresses kiwi PMEI. Moreover, the overexpression of AtPMEI-2 in Arabidopsis results in a substantial reduction in its susceptibility to the Turnip vein-clearing virus (Lionetti et al., 2014).

PMEI was first discovered in the kiwi fruit (Balestrieri et al., 1990), and later detected in other plants, including Arabidopsis, pepper, and tomato (Solanum lycopersicum; Raiola et al., 2004; An et al., 2008; Reca et al., 2012). Disulfide bond formation and ionic interactions may be important for PMEI stability. In particular, subtle pH changes in the microenvironment greatly impact the stability of partially buried ionic interactions (Bonavita et al., 2016). For instance, the interaction between the tomato PME and the kiwi PMEI grows rapidly weaker with an increase in pH, and no complex is observed at pH 8.0 (Jolie et al., 2010). Further, at a pH >7.0, the inhibition of banana (Musa nana), carrot (Daucus carota), and strawberry (Fragaria × ananassa) PMEs by the kiwi PMEI is also reduced (D’Avino et al., 2003). Since the enzymatic activity of PME is regulated by pH, the stability of the PMEI-PME interaction is also affected by pH (Denès et al., 2000).

Cotton is an important cash crop worldwide, and is widely cultivated for the economic value of its fibers (Xu et al., 2011). The cotton fiber is an ideal model for plant cell elongation and cell wall biogenesis studies, because of its highly elongated structure (Kim et al., 2001). Cotton Verticillium wilt is caused by Verticillium dahliae, a soil-borne plant pathogenic fungus. The disease is difficult to control in cotton as the hyphae reside in the vascular (xylem) tissues of the plant. Moreover, this pathogen can overwinter as a mycelium within perennial hosts or in tubers, bulbs, or seeds, as well as in other propagative organs of the plant. Moreover, resting structures known as microsclerotia support the durable vegetative resting structures of Verticillium, which survive in the soil for many years (Fradin and Thomma, 2006). Therefore, Verticillium wilt leads to severe cotton yield loss each year and represents a major concern for cotton producers (Gao et al., 2011). However, the physiology of plant resistance against Verticillium is largely unexplored. Moreover, even less is known about the crucial fungal components involved in its pathogenicity. Historically, the roles of cell wall degrading enzymes, such as endopolygalacturonase, have attracted much attention (Fradin and Thomma, 2006). Recently, the Ve locus has been bred in various tomato cultivars and was shown to be responsible for resistance to V. dahliae (Kawchuk et al., 2001). Nevertheless, not much is known about the genetic and molecular mechanisms that underlie cotton resistance to Verticillium infection.

In the current study, we investigated whether cotton GhPMEI3 is active against GhPME2 (a type I enzyme) and GhPME31 (a type II enzyme). To this end, we expressed and purified the three proteins and performed in vitro inhibition studies. Enzyme inhibition assays and a PMEI-PME interaction analysis indicated that GhPMEI3 very efficiently inhibits these cotton PMEs. Furthermore, heterologous expression of GhPMEI3 in Arabidopsis lowered pectin levels in the cell wall, markedly enhancing the plant’s resistance to the V. dahliae fungal pathogen. In silico protein modeling, docking, and electrostatic charge distribution analyses were also performed to clarify the structural basis of the PMEI-PME interaction. The current study highlights the regulation of the PMEI-PME interaction and pectin deesterification in plants. Since fungal pathogens are unable to utilize methyl esterified pectin, and this modification reduces the ability of other cell wall-degrading enzymes to hydrolyze pectin, the PMEI-PME interaction enhances plant resistance to pathogens.

RESULTS

Bioinformatics Analysis of GhPMEI3, GhPME2, and GhPME31

The inferred domain architecture of GhPMEI3 was characterized using multiple alignment and bioinformatics approaches. GhPMEI3 was predicted to be encoded by a 720-bp open reading frame encoding a 240-amino acid protein with no signal peptide. The theoretical M r of GhPMEI3 is 25.96 kD (that of the purified protein was 43.96 kD, with the protein linked to an 18-kD Trx tag, S tag, and His tag), with a pI of 5.07. The M rs of GhPME2 and GhPME31 are 82.96 kD and 61.53 kD (including a 26-kD GST-tag from the vector), with isoelectric points of 9.23 and 7.52, respectively.

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To functionally characterize GhPMEI3, multiple sequence alignment with various PMEIs was performed (Fig. 1). Similarly to other PMEIs, the structure of GhPMEI3 is mainly helical, with four long antiparallel helices (α1–4) arranged in a classic up-and-down four-helical bundle (Fig. 1; Di Matteo et al., 2005). GhPMEI3 has four Cys residues that form two disulfide bridges, one of which connects the α2 and α3 helices, stabilizing the interior of the bundle. The other disulfide bridge connects helices αa and αb in the N-terminal region (Di Matteo et al., 2005). PMEIs belong to the PF04043 family (Pfam database, http://pfam.xfam.org/) and share several structural properties with INH; however, their target enzymes are not related (Di Matteo et al., 2005; Lionetti et al., 2015).