Studies on the plant immune system involving the PAMP receptor RLP23 in Arabidopsis thaliana

概要

シロイヌナズナの PAMP 受容体 RLP23 が関与する植物免疫機構に関する研究
大野 恵梨佳

CHAPTER I. Plant immune responses are quantitatively different between nlp24induced immunity and flg22-induced immunity

Plants recognize conserved microbial molecules, referred to as pathogen-associated
molecular patterns (PAMPs), and activate immunity. In Arabidopsis thaliana, immune
responses are induced by two known PAMPs, nlp24 derived from secreted proteins
termed necrosis- and ethylene-inducing-like proteins (NLPs), conserved in a broad range
of fungi, bacteria and oomycetes, and flg22 derived from bacterial flagellin. Here, I found
that there were quantitative differences between nlp24- and flg22-induced immune
responses. The expression of PAD3, a gene required for camalexin synthesis, and ERF1,
a gene responsive to ethylene, were strongly induced by nlp24 treatment in comparison
to flg22. Moreover, the timing of gene expression for these genes were also distinct
between nlp24- and flg22-induced immunity in A. thaliana. Conversely, the reactive
oxygen species (ROS) production and MAPK activation were more induced by flg22 than
nlp24. The flg22 peptide is recognized by the leucine-rich repeat receptor kinase (LRRRK) type receptor FLAGELLIN-SENSITIVE 2 (FLS2), while the nlp24 peptide is
recognized by the leucine-rich repeat receptor protein (LRR-RP) type receptor RLP23
that lacks a kinase domain in the intracellular region. Complemented Arabidopsis rlp23
mutant with chimeric receptors having the intracellular kinase domain of FLS2 and the
extracellular LRR domain of RLP23 exhibited the ROS production after nlp24 treatment.
However, the amount of ROS production observed in these transgenic plants treated with

nlp24 was not comparable to that in wild-type Arabidopsis treated with flg22, and was
even lower than that in transgenic plants harboring full-length RLP23. These results
suggest that the structural differences in the pattern-recognition receptors are insufficient
to fully explain the molecular mechanisms underlying the differences between nlp24induced immunity and flg22-induced immunity.

CHAPTER II. Arabidopsis BAK1 plays distinct roles between nlp24-induced immunity
and flg22-induced immunity

During PAMP-induced immunity, pattern recognition receptors engage with their
corresponding co-receptors to mediate downstream signaling. FLS2 interacts with its coreceptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE
1 (BAK1) immediately following flg22 treatment. On the other hand, RLP23 interacts
with BAK1 upon nlp24 treatment and also constitutively interacts with another coreceptor SUPPRESSOR OF BIR1-1 (SOBIR1). I revealed that nlp24-induced immunity
involves both BAK1-dependent and BAK1-non-essential pathways, whereas flg22induced immunity largely relies on BAK1. All tested immune responses, including ROS
production, expression of WRKY33 and PAD3 expression, and MAPK activation, were
diminished in the double mutants of BAK1 and its homolog BAK1-LIKE 1 (BKK1)
following flg22 treatment. However, the nlp24 treatment induced MAPK activation and
the expression of WRKY33 and PAD3 in the bak1-5 bkk1 mutant, although the immune
responses were not intact. The ROS production was not induced in bak1-5 bkk1 after
nlp24 treatment. In contrast, the sobir1-12 mutant failed to activate all the tested immune
responses after nlp24 treatment, while it retained the induction of the immune responses

following flg22 treatment to the same extent as wild-type Col-0. Moreover, the
pretreatment with nlp24 resulted in a reduction of the growth of a bacterial pathogen
Pseudomonas syringae pv. tomato DC3000 in the bak1-5 bkk1 mutant. This reduction
was not observed in pad3 mutant pretreated with nlp24. These results indicate the
presence of a BAK1-non-essential pathway in addition to the BAK1-dependent pathway
in nlp24-induced immunity, and suggest that BAK1 is involved in the differences
between nlp24-induced immunity and flg22-induced immunity.

CHAPTER III. RLP23 is required for Arabidopsis immunity against the grey mould
pathogen Botrytis cinerea

The flg22 receptor FLS2 has been reported to be essential for the resistance to
bacterial pathogen P. syringae pv. tomato DC3000. However, the actual contribution of
nlp24 receptor RLP23 to plant immunity against pathogens is still unclear. Here, I
revealed that RLP23 was required for Arabidopsis immunity against the necrotrophic
fungal pathogen Botrytis cinerea. In contrast, RLP23 was dispensable for the immunity
against the necrotrophic fungus Alternaria brassicicola and the hemi-biotrophic fungus
Colletotrichum higginsianum. Interestingly, microscopic observation revealed that the
invasion ratio of B. cinerea was increased in the rlp23 mutants compared with the wildtype plants.

B. cinerea

possesses

two

NLP

genes,

named

BcNEP1 and

BcNEP2. BcNEP1 was preferentially expressed before/during invasion, in contrast to the
BcNEP2 that was expressed at the late infection phase. The nlp sequences derived from
BcNEP1 as well as BcNEP2 were recognized by Arabidopsis RLP23, although the nlp
sequence of BcNEP1 contained three additional amino acids that were absent in BcNEP2

and other NLPs, such as the NLPs of C. higginsianum, Phytophthora parasitica
(oomycete) and Bacillus subtilis (bacterium). I found that A. brassicicola expressed
its NLP gene (AbNLP1) preferentially at the late infection phase, rather than
before/during pathogen invasion. I also found that the co-inoculation of A. brassicicola
with the synthetic nlp peptide from BcNEP1 strengthened the immunity against A.
brassicicola. Collectively, these results revealed that RLP23 contributes to
the Arabidopsis pre-invasive resistance to B. cinerea via the recognition of the NLP
proteins, mainly BcNEP1, which are secreted at the early infection phase. ...