Disease resistance to Pectobacterium carotovorum is negatively modulated by the Arabidopsis Lectin Receptor Kinase LecRK-V.5

Abstract

Plant stomata function in disease resistance by restricting bacteria entry inside leaves. During plant-bacteria interactions, stomatal closure is initiated by the recognition of Microbe-Associated Molecular Patterns (MAMPs). Recently, we have shown that the Lectin Receptor Kinase V.5 (LecRK-V.5) negatively regulates bacterium- and MAMP-induced stomatal closure upstream of Reactive Oxygen Species (ROS) production mediated by abscisic acid signaling. Closed stomata in lecrk-V.5 mutants are correlated with constitutive high level of ROS in guard cells. Consequently, lecrk-V.5 mutants are more resistant to hemi-biotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000). In this report, we further investigate the role of LecRK-V.5 in resistance against necrotrophic bacteria Pectobacterium carotovorum ssp. carotovorum (Pcc). Upon surface-inoculation lecrk-V.5 mutants exhibited enhanced resistance against Pcc whereas a wild-type level of resistance was observed using infiltration-inoculation, an inoculation method that bypasses the epidermal barrier. Enhanced resistance of dip-inoculated lecrk-V.5 mutants against necrotrophic bacteria, that induce different defense responses than hemi-biotrophic bacteria, further suggests a possible role for LecRK-V.5 in stomatal immunity.

Stomatal closure as part of the Pattern-Triggered Immunity (PTI) response is one of the first lines of plant defense against bacterial invasion.¹ Pseudomonas syringae pv tomato DC3000 (Pst DC3000), Xanthomonas campestris and Escherichia coli bacteria indeed induce stomatal closure in Arabidopsis within 1 to 2 h post inoculation.^2,3 Pst DC3000 is able to reopen stomata 3 to 4 h after infection through the action of the virulence factor coronatine (COR).³ Recently, a genetic screen identified several susceptible to coronatine-deficient (scord) mutants defective in stomatal closure upon COR-deficient Pst DC3000 inoculation and two of them showed wild-type (WT) apoplastic defense.⁴ The promotion of stomatal closure by bacteria is mediated by the perception of Microbe-Associated Molecular Patterns (MAMPs) such as the flagellin peptide 22 (flg22).³ Flagellin Insensitive 2 (FLS2) is required for flg22-induced stomatal closure and plays an essential role in stomatal closure mediated by Pst DC3000.^3,5

We have recently identified the Lectin Receptor Kinase V.5 (LecRK-V.5) as an important negative regulator of stomatal immunity.⁶ LecRK-V.5 expression is rapidly induced in guard cells after flg22 and Pst DC3000 treatments. Overexpression of LecRK-V.5 leads to defects in MAMP-induced stomatal closure and Reactive Oxygen Species (ROS) production in guard cells.⁶ By contrast, lecrk-V.5 mutants exhibit constitutive stomatal closure that is correlated with a high level of ROS in stomata. Upon PTI activation, lecrk-V.5 mutants are insensitive to COR-mediated stomatal reopening and lecrk-V.5 stomata are still closed at 3 h post inoculation with Pst DC3000.⁶ As a biological consequence, lecrk-V.5 mutants are more resistant than WT to Pst DC3000 dip-inoculation. However, using infiltration inoculation, no difference in symptoms and in bacterial growth were observed between mutants and WT plants indicating that disease resistance is linked to stomatal defense.⁶ By contrast, LecRK-V.5 overexpression (OE) lines are more susceptible than WT plants to Pst DC3000 dip-inoculation probably because of faster stomatal reopening.⁶

LecRK-V.5 Negatively Regulates Disease Resistance to the Necrotrophic Bacteria Pectobacterium carotovorum ssp. carotovorum

To evaluate the possible contribution of LecRK-V.5 to disease resistance against necrotrophic bacteria, that activate a different Arabidopsis defense response than hemi-biotrophic Pst DC3000,⁷ we first analyzed LecRK-V.5 expression after Pectobacterium carotovorum ssp. carotovorum (formerly Erwinia carotovora ssp. carotovora) strain WPP14 (Pcc) infection.⁸ Analysis of LecRK-V.5 expression by qRT-PCR⁶ revealed that LecRK-V.5 was induced more than 10-fold at 1 d after Pcc dip-inoculation (Fig. 1A). We also dip-inoculated lecrk-V.5 mutants with Pcc and quantified in planta bacterial growth. At 1 d post Pcc dip-inoculation, growth of bacteria in lecrk-V.5–1 and lecrk-V.5–2 mutants was about 5 to 10-fold lower than in WT control plants (Fig. 1B), indicating that both lecrk-V.5 mutants were resistant to Pcc. When bacteria were directly infiltrated into the apoplast, lecrk-V.5 mutants did not show any significant differences in bacterial titers when compared with WT control plants (Fig. 1C). These results suggest that enhanced resistance of lecrk-V.5 mutants to Pcc is not linked to an increase in apoplastic defense but is likely due to constitutive stomatal closure, confirming our observations with Pst DC3000.⁶ By contrast, lines overexpressing LecRK-V.5 exhibited enhanced susceptibility to Pcc dip-inoculation with bacterial titers about 10-fold higher than WT plants (Fig. 1D). Lines overexpressing LecRK-V.5 are therefore more susceptible to both Pcc and Pst DC3000.⁶ Collectively, these results indicate that LecRK-V.5 negatively regulates Arabidopsis resistance to both hemi-biotrophic and necrotrophic bacteria, possibly through negatively regulating Arabidopsis stomatal immunity.

Figure 1. A negative role for LecRK-V.5 in Arabidopsis resistance to Pcc. (A) Analysis of LecRK-V.5 expression by qRT-PCR in Arabidopsis thaliana (Ler ecotype) at 0 and 1 d after dip-inoculation with 1 × 10⁵ cfu.ml⁻¹Pectobacterium carotovorum (Pcc) strain WPP14. Expression level was normalized to EF-1 and compared with time 0 with a defined expression value of 1. Data represent average values ± SE (n = 3). (B) Bacterial growth in lecrk-V.5–1 and lecrk-V.5–2 mutants and corresponding Ler and Col-0 WT plants at 1 d after dip-inoculation with 1 × 10⁵ cfu.ml⁻¹Pcc. (C) Bacterial growth in lecrk-V.5 mutants and WT (Ler and Col-0) infiltrated-inoculated with 1 × 10⁴ cfu.ml⁻¹Pcc. (D) Bacterial growth assessed at 1 d after dip-inoculation with Pcc in WT (Col-0) and LecRK-V.5 overexpression lines (OE-1 and OE-2). Bacterial quantification was determined as described earlier.^6,11 Data represent average values ± SD. Statistical differences between WT controls and mutant or plants overexpressing LecRK-V.5 are detected with a t-test (p < 0.01, n = 6). All experiments were repeated at least twice with similar results. dpi, day post inoculation; cfu, colony forming units.

Conclusions

An effective stomatal closure upon MAMPs perception is critical for plant resistance to bacteria.¹ We showed that LecRK-V.5 is a negative regulator of MAMP-triggered stomatal closure.⁶ In this report we demonstrate that LecRK-V.5 represses resistance to necrotrophic Pcc bacteria possibly through action on stomatal immunity. Similar divergent phenotypes after surface- or infiltration-inoculation were previously observed using the hemi-biotrophic bacteria Pst DC3000.⁶ Moreover, a recent study using mutant and overexpression lines of LecRK-VI.2 suggested the importance of stomatal immunity in resistance against both Pcc and Pst DC3000 infection.⁹ Necrotrophic and hemi-biotrophic pathogens have different lifestyles that induce a different set of defense responses in Arabidopsis.⁷ However, as the first barrier against microbes, stomatal immunity is important for plant defense against pathogenic and non-pathogenic bacteria that penetrate into leaves via natural opening.¹⁰ At later stage of infection, other PTI responses occur preferentially in mesophyll cells.^4,5 This study provides additional evidences that upon MAMP perception stomatal immunity is effective against a wide range of pathogenic bacteria with different lifestyles.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.