Elsevier

Gene

Volume 753, 30 August 2020, 144802
Gene

Research paper
An Arabidopsis DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 is required for resistance against various phytopathogens and tolerance to salt stress

https://doi.org/10.1016/j.gene.2020.144802Get rights and content

Highlights

  • Lipid transfer proteins have important implications in plant and animal defense responses.

  • DRN1 is a putative nsLTP which is strongly suppressed by bacteria, ROS (O2radical dot), and salt stress.

  • DRN1 suppression depends on a pathogen derived factor that requires a functional TTSS.

  • DRN1 is targeted by a novel miRNA DmiR that is specifically induced by pathogens and ROS.

  • DRN1 is required for resistance against Pseudomonas, Botrytis, and tolerance to salt stress.

Abstract

Synchronous and timely regulation of multiple genes results in an effective defense response that decides the fate of the host when challenged with pathogens or unexpected changes in environmental conditions. One such gene, which is downregulated in response to multiple bacterial pathogens, is a putative nonspecific lipid transfer protein (nsLTP) of unknown function that we have named DISEASE RELATED NONSPECIFIC LIPID TRANSFER PROTEIN 1 (DRN1). We show that upon pathogen challenge, DRN1 is strongly downregulated, while a putative DRN1-targeting novel microRNA (miRNA) named DRN1 Regulating miRNA (DmiR) is reciprocally upregulated. Furthermore, we provide evidence that DRN1 is required for defense against bacterial and fungal pathogens as well as for normal seedling growth under salinity stress. Although nsLTP family members from different plant species are known to be a significant source of food allergens and are often associated with antimicrobial properties, our knowledge on the biological functions and regulation of this gene family is limited. Our current work not only sheds light on the mechanism of regulation but also helps in the functional characterization of DRN1, a putative nsLTP family member of hitherto unknown function.

Introduction

The interaction between plants and microbes is not always a beneficial relationship, as evidenced by the diseases caused by various phytopathogens. Thus, plants have developed an elaborate and multilayered defense strategy to detect and combat such challenges from harmful microbes. Primary barriers include preformed defenses like glandular trichomes, epidermal hairs, waxy cuticle layer, production of antimicrobial secondary metabolites (including antifungal and antibacterial compounds), and cell wall strengthening mechanisms like lignification and callose deposition (Agrios, 2005, De Wit, 2007). These defense mechanisms are widely distributed across the plant kingdom. Additionally, plants have two levels of induced defenses against pathogens. The first level of defense, commonly known as basal defense, is usually activated by the perception of pathogen-associated molecular patterns (PAMPs). PAMPs are highly conserved molecules, such as elongation factor Tu (EF-Tu), flagellin, chitin, nep1-like protein (nlp10), lipo-chitooligosaccharides (LCO), lipopolysaccharide, peptidoglycan, oligogalacturonide etc. that are integral to the survival of the pathogen (Boller and Felix, 2009, Macho and Zipfel, 2014, Zipfel and Oldroyd, 2017). Plants can sense these PAMPs via receptors on the surface of the plasma membrane to initiate PAMP-triggered immunity (PTI), which in turn leads to activation of several defense genes via mitogen-activated protein (MAP) kinase signaling leading to basal resistance including oxidative burst at the site of contact (Lamb and Dixon, 1997, Li et al., 2005, Macho and Zipfel, 2014, Mundt, 2014). However, due to pathogen adaptations to overcome such host surveillance by incorporating rapidly evolving effectors and modified infection strategies, basal defense on its own may not be enough to prevent pathogen growth (Chisholm et al., 2006). Nonetheless, basal defense does contribute towards limiting the spread of the pathogen while providing durable resistance in the long term (Torres, 2010, Zhang and Coaker, 2017).

The second layer of defense in plants consists of highly evolved effector-based signaling mechanisms that help them recognize and respond to the presence of very specific pathogen effectors and mount a precise defense response (Abramovitch and Martin, 2004, Nishimura and Dangl, 2010). Throughout evolution, pathogens have developed mechanisms to suppress plant defense responses by secreting effector proteins with very specialized functions to help them overcome plant defenses (Chisholm et al., 2006, Cunha et al., 2007, Chiang and Coaker, 2015). In many cases, these effector molecules are delivered directly inside the host cells to suppress plant defenses through a pathogen-encoded delivery apparatus (Yuan and He, 1996, Collmer et al., 2000, Abramovitch and Martin, 2004). Once inside the host, these effector proteins have a host target, disruption of which may include but is not limited to killing plant cells which releases the nutrients for pathogens to use for their expansion and growth on the host (Cunha et al., 2007, Kim et al., 2008). In response to the effector proteins of the pathogens, plants have evolved cognate Resistance (R) genes with precise functions to detect the presence of the pathogen effector protein and induce a rapid yet robust defense response known as effector-triggered immunity (ETI), resulting in the elimination of the pathogen or limiting its spread (Chisholm et al., 2006). One of the most important outcomes of ETI is the activation of hypersensitive response (HR). HR is a form of programmed cell death (PCD) of host tissue induced at the site of infection. Activation of HR is accompanied by activation of downstream defense genes, a rapid burst of reactive oxygen species (ROS), changes in ion fluxes and protein phosphorylation, and other responses, all of which eventually lead to the death of the infected cells and elimination of the pathogen at the site of infection (Jones and Dangl, 2006, Kim et al., 2008, Chiang and Coaker, 2015, Cui et al., 2015). In most cases, cell death following HR or disease caused by the pathogen leads to activation of a long-lasting and broad-spectrum resistance termed as the systemic acquired resistance (SAR). Plants that are defective in the accumulation of salicylic acid (SA) or signaling mediated by SA are unable to activate SAR (Durrant and Dong, 2004).

Earlier work from multiple groups focused on a genome-wide analysis for differentially regulated genes during biotic stress (including in the model plant Arabidopsis thaliana) in response to various pathogens (Schenk et al., 2000, Tao et al., 2003, De Vos et al., 2005, Eulgem, 2005, Thilmony et al., 2006, Atkinson et al., 2013, Sham et al., 2015). Such studies have helped shed light not only on host genes that are upregulated to fight pathogen infection but also those genes that are actively downregulated after pathogen treatment (Kourelis and van der Hoorn, 2018). A whole-genome microarray analysis undertaken as a part of separate study in our lab to identify differentially expressed genes in response to biotic stress led to the identification of a putative nonspecific lipid transfer protein (nsLTP) encoded by AT2G45180, as a candidate gene that was strongly suppressed in response to avirulent bacterial phytopathogen Pseudomonas syringae pv. tomato DC3000 (avrRpm1) (S. Maqbool, personal communication).

In the work presented here, we have characterized AT2G45180 of previously unknown function and have annotated it as Disease Related Nonspecific Lipid Transfer Protein 1 (DRN1) based on its regulation by pathogens and its requirement for proper defense response by the host. A quick assessment of The Arabidopsis Information Resource (TAIR) indicates that DRN1 shows similarity to the nsLTP family of genes. The nsLTP family consists of low molecular weight basic proteins (Kader, 1996). One of the characteristic features shared by all nsLTP family members is the consensus motif of eight cysteine residues (8CM/ECM) that make four disulfide bridges forming an a-helix with a hydrophobic cavity or tunnel, which can bind and transport different lipids and other hydrophobic molecules (Kader, 1996, Arondel et al., 2000, Buhot et al., 2001, Cheng et al., 2004, Yeats and Rose, 2008). LTPs have been shown to be involved in a variety of biological processes such as seed germination, reproduction, vegetative and developmental growth, adaptation to stress, symbiosis, defense, cryoprotection etc. (Hincha et al., 2001, Carvalho and Gomes, 2007, Lascombe et al., 2008, Wang et al., 2009, Pii et al., 2009, Chae et al., 2010, Petti et al., 2010, Safi et al., 2015, Finkina et al., 2016, Jülke and Ludwig-Müller, 2016), and are often associated with surface and cuticular wax in plants (Pyee et al., 1994, Liu et al., 2014, Pan et al., 2016). Finally, many of the nsLTPs that are involved in defense signaling against pathogens have been shown to either possess direct antimicrobial activities in vitro (Segura et al., 1993, Cammue et al., 1995, Wang et al., 2004, Gonorazky et al., 2005, Roy-Barman et al., 2006, Sun et al., 2008, Edstam et al., 2013) or are required for proper defense response (García-Olmedo et al., 1995, Maldonado et al., 2002, Ho et al., 2005, Salcedo et al., 2007, Sarowar et al., 2009, Zhu et al., 2012, Safi et al., 2015, Jülke and Ludwig-Müller, 2016). In general, nsLTPs have been suggested to transfer lipid molecules that may play an important role in fatty acid-mediated defense signaling by forming a sterol-elicitin complex. This complex is perceived by the receptors on the plasma membrane. Recombinant tobacco LTP1 was shown to bind jasmonic acid (JA), and the resulting complex (LTP1-JA) was shown to bind to an elicitin receptor and induce long-distance systemic resistance against the fungal pathogen Peronospora parasitica (Blein et al., 2002, Buhot et al., 2004). In addition, some of the constitutively expressed nsLTPs have been reported to localize in the apoplast where they may help boost basal immunity as the first line of defense against pathogens (Cheng et al., 2004).

Further relevance of work to understand the regulation of this important class of genes comes from the fact that LTPs not only play a role in plant defense but also have an unexpected role in animal defense responses. This class of proteins is additionally a major type of allergen found not only in pollen but also in fruits, vegetables, and cereals from various plant genera that are relatively stable proteins and are resistant to thermal and chemical denaturation and enzymatic digestion (Salcedo et al., 2004, Breiteneder and Mills, 2005, Zuidmeer and Van Ree, 2007, Egger et al., 2010, Pascal et al., 2012, Van Winkle and Chang, 2014, Asero et al., 2018). Though nsLTP family members seem to be excellent candidates to bolster plant defense (Salcedo et al., 2004), their application without a comprehensive understanding of their role and regulation could lead to unintended health consequences for both plants and humans. In our current study, we hypothesized that DRN1 expression is altered by pathogen challenge and tested if this alteration led to any functional significance associated with this nsLTP family-related gene. Furthermore, we propose that a novel microRNA (miRNA)-mediated regulation mechanism underlies the post-transcriptional regulation of this gene in response to bacterial pathogens.

Section snippets

Plant materials and growth conditions

All Arabidopsis thaliana plants used were of Colombia (Col-0) ecotype unless mentioned otherwise. Plants were grown in soil (Metro-Mix 360, Sun Gro Horticulture) or on plates containing ½-strength Murashige and Skoog (½-MS) media (pH 5.8, 0.8% agar) supplemented with appropriate antibiotics. Plant growth rooms were kept at 22/20 °C (day/night), 50–60% relative humidity, and a photosynthetic photon flux density (PPFD) of 75–100 μmol m−2sec−1 with a 10 h (L)/14 h (D) photoperiod for short day

DRN1 is a nsLTP family-related gene that is strongly expressed in photosynthetically active tissues

The putative nsLTP protein DRN1 is a 13.9 kDa basic protein with the characteristic 8CM/ECM that is typical of this relatively uncharacterized family of proteins. Moreover, since DRN1 is annotated as a putative nsLTP on TAIR, we performed a search on TAIR for candidate “lipid transfer protein” in Arabidopsis. Phylogenetic analysis was performed using Multalin (Corpet, 1988) (Fig. 1A) and ClustalW (Madeira et al., 2019) (Supplementary Fig. 1). Furthermore, we added the well-characterized

Discussion

We have discovered that the previously uncharacterized nsLTP family-related DRN1 is not only suppressed in response to multiple biotic and abiotic stresses but is also a positive regulator of plant defense responses. Our gene expression analysis revealed that DRN1 is suppressed strongly in response to bacterial pathogens (Pst DC3000 and Psm ES4326). This pathogen-mediated suppression of DRN1 is dependent on a heat-labile pathogen-derived factor that is dependent on the bacterial TTSS for

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors acknowledge assistance from Dr. Timothy J. Fokken, Dr. Alyssa C. Lau, and other members of the Raina Lab for their help with the maintenance of plants and pathogens used and some plant data collection. We also thank Dr. Allan Collmer for Pst DC3000 (hrcC) and Pst DC3000 (hrcU), and Dr. Greg Martin for P. syringae pv. tabaci. We would like to thank Drs. Jeff Dangl, Jean Greenberg, Xinnian Dong, and Mary Wildermuth for lsd1, acd2, npr1-1, and eds1-16 seeds, respectively.

Author contributions

N.D. and R.R. planned and designed experiments; N.D., J.C.S., and, I.E. performed experiments and data analysis; N.D., J.C.S., and, I.E., and R.R. analyzed the data; N.D. prepared the original draft; N.D., J.C.S., I.E., and R.R. reviewed, edited, and finalized the manuscript.

Funding

Work in the laboratory of R.R. was supported by a grant from the National Science Foundation (IOS-1146128).

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