The Arabidopsis LECTIN RECEPTOR KINASE-VI.2 is a functional protein kinase and is dispensable for basal resistance to Botrytis cinerea

Abstract

Sensing of microbial pathogens by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) elicits a defense program known as PAMP-triggered immunity (PTI). Recently, we have shown that the Arabidopsis thaliana L-TYPE LECTIN RECEPTOR KINASE-VI.2 (LecRK-VI.2) positively regulates bacterial PTI. In this report, we suggest by in silico analysis that the kinase domain of LecRK-VI.2 is functional. LecRK-VI.2 also demonstrated auto-phosphorylation activity in vitro in the presence of divalent metal cations indicating that LecRK-VI.2 has the ability to auto-phosphorylate. We further investigate the role of LecRK-VI.2 in Arabidopsis resistance to the necrotrophic fungal pathogen Botrytis cinerea. Disruption of LecRK-VI.2 did not affect Arabidopsis resistance to B. cinerea. Accordingly, wild-type upregulation levels of PTI-responsive WRKY53, FRK1, NHL10, CYP81F2 and CBP60 g after treatment with the fungal PAMP chitin were observed in lecrk-VI.2-1. These data provide evidences that the kinase domain of LecRK-VI.2 is active and show that LecRK-VI.2 is not critical for resistance to the fungal pathogen B. cinerea.

In plants, defense responses are induced by pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs). This recognition activates a defense program known as PAMP-triggered immunity (PTI).¹ Arabidopsis defense responses against necrotrophs such as B. cinerea are mediated by jasmonic acid (JA) and/or ethylene (ET) signaling cascades.² Arabidopsis resistance to B. cinerea is also salicylic acid (SA)-dependent.^3,4 Chitin is a major fungal PAMP that triggers the PTI defense response in plants.⁵ Chitin is produced by B. cinerea and is recognized by CERK1 PRR (chitin elicitor receptor kinase, also known as LysM-RLK1).⁵ Binding of PAMPs to extracellular domains of receptor-like kinases (RLKs) is thought to activate the intracellular kinase domains of RLKs.⁶ The lectin receptor kinases are RLKs characterized by the presence of an extracellular legume lectin-like domain, a transmembrane domain and an intracellular serine/threonine (Ser/Thr) kinase (STK) domain. Lectin receptor kinases are divided in three types, G, C, and L based on their extracellular lectin motif.⁷ Recently, we demonstrated that the L-type lectin receptor kinase VI.2 (LecRK-VI.2) positively regulates Arabidopsis bacteria-mediated PTI.⁸ Here we suggest that LecRK-VI.2 possesses a functional kinase domain that is able to auto-phosphorylate. In addition, resistance to the necrotrophic fungal pathogen B. cinerea and expression of PTI-responsive genes after chitin treatment were at wild-type levels in a lecrk-VI.2-1 T-DNA insertion mutant. Our results suggest that LecRK-VI.2 specifically regulate bacteria-mediated PTI.

LecRKVI.2 is a Functional Kinase Protein and is not Essential for Basal Resistance to Botrytis cinerea

In order to evaluate the functionality of the LecRK-VI.2 kinase domain in silico, amino acid sequences of kinase domain of various published LecRKs such as LecRK-V.1, LecRK-VII.1, and LecRK-VII.2,⁷ PsLecRLK⁹ and OsSIK1¹⁰ were aligned with the kinase domain (KD) of LecRK-VI.2. We found that the amino acids reported to be essential for catalytic activities of all 11 kinase sub-domains¹¹ (numbered from I-XI, Fig. 1A) from Arabidopsis, pea and rice were highly conserved in LecRK-VI.2, suggesting that its kinase domain is functional. LecRK-VI.2 exhibited divalent metal cations dependent auto-phosphorylation activity in vitro (Fig. 1B). To evaluate Arabidopsis LecRK-VI.2 possible role in the resistance response to other types of pathogens, we inoculated the lecrk-VI.2-1 mutant with the necrotrophic fungus B. cinerea. The mutant lecrk-VI.2-1 demonstrated a wild type resistance response to the necrotroph B. cinerea (Fig. 1C). In addition, no significant differences in expression levels of PTI responsive genes between the mutant lecrk-VI.2-1 and the wild-type Col-0 control were observed after chitin treatment (Fig. 1D), suggesting that PTI activation after treatment with the fungal PAMP chitin is not dependent on a functional LecRK-VI.2. Together, these observations suggest that LecRK-VI.2 is not critical for Arabidopsis resistance to fungal pathogens.

Figure 1.LecRKVI.2 is a functional protein kinase and is dispensable for Arabidopsis resistance to B. cinerea. (A) Alignment comparison of the predicted amino acid sequences of LecRK-VI.2 with 3 Arabidopsis LecRKs (LecRK-V.1, LecRK-VII.1 and LecRK-VII.2), the rice OsSIK1 and the pea PsLecRLK. Sequences above the Roman numerals in black boxes indicate the 11 sub-domains characteristic of a typical protein kinase. (B) Expression and purification of LecRK-VI.2-KD from E.coli using affinity resin and phosphorylation assay. The GST-fusion protein was stained with Coomassie blue and confirmed by peptide sequencing. GST was used as a control (left panel). GST fusion protein LecRKVI.2-KD (2 µg) was incubated with ATP for 30 min in the presence of 5 mM MnCl₂, 5 mM CaCl₂ or 5 mM MgCl₂. Phosphorylation signal was observed with ProQ Diamond phosphoprotein gel staining (right panel). (C) B. cinerea disease symptoms. Arabidopsis leaves were droplet-inoculated and symptoms were visualized 2 d later. Experiments were repeated 3 times with similar results. Bar = 1 cm. Error bars are SD (n = 18 leaves). Different letters indicate statistically significant differences compared with the wild-type Col-0 (LSD test; p < 0.05). (D) Relative expression levels of WRKY53, FRK1, NHL10, CYP81F2 and CBP60 g were analyzed 30 and 60 min after chitin infiltration (50 µg/ml). EF-1 and UBQ10 were used for normalization. Relative gene expression levels were compared with buffer control (defined value of 1) by qRT-PCR analyses. The values are the means ± SD of three biological replicates (n = 9). No significant differences to wild-type Col-0 were observed when based on a t-test (p < 0.01).

Conclusions

We showed that LecRKVI.2 is a positive regulator of bacterial PTI response in Arabidopsis.⁸ In this report we demonstrate that LecRKVI.2 possesses a functional kinase domain. PTI, SA- and JA-mediated signaling cascades are intimately bound¹ and manipulation of PTI through alteration of LecRK-VI.2 expression may alter JA signaling and resistance to necrotrophic fungi. We therefore asked whether LecRKVI.2 plays a role in resistance to a necrotrophic fungal pathogen such as B. cinerea. Resistance to B. cinerea and upregulation of PTI-responsive genes after treatment with the fungal PAMP chitin were at wild-type levels in lecrk-VI.2-1. Similarly, the PTI response triggered by chitin does not depend on BAK1.¹² Collectively these observations indicate that LecRK-VI.2 is not required for basal resistance to B. cinerea and suggest that LecRK-VI.2 is specifically involved in bacterial defense signaling. LecRK-VI.2 may thus function as a positive regulator specific for bacteria-triggered PTI.