Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread

Abstract

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system.

Keywords: BED domain; decoy; genome; nucleotide-binding and leucine-rich repeat domain (NLR); plant immunity.

Figure 1

Examples of integration of unusual domains in nucleotide‐binding and leucine‐rich repeat (NLR) proteins in plants. Protein domains of the different NLRs were established by an InterPro search (http://www.ebi.ac.uk/interpro/) or NCBI domain search (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) along the full‐length protein (black line). The Interpro search does not detect coil–coil domains often found in NLRs, thus explaining their absence here. Canonical NLR domains (Tol Interleukin Receptor (TIR), P‐loop/NB‐ARC and LRR) are indicated in pink and green. Although these domains may be larger, the detection by the Interpro search only indicates the borders defined by the Interpro domains. (a) The R3a, R1 and KR1 cloned resistance proteins contain unusual protein domains besides the canonical NLR structure. In KR1, IPR011991 represents a winged helix‐turn‐helix DNA‐binding domain, whereas FAM75 and DUF3542 are domains of unknown function. (b) The WRKY domain (brown) was integrated several times in plant NLR genes from different species and some examples are shown here. An additional NAC domain is also found in the sorghum protein. (c) The Arabidopsis thaliana NLR At4g12020 is highly modular and contains three types of additional domains besides the canonical TIR‐NBS‐LRR domains: a WRKY domain, a PAH domain of unknown function (IPR002832) and a kinase‐like domain (IPR011009).

Figure 2

Most plant species show unusual domains in their nucleotide‐binding and leucine‐rich repeat (NLR) proteins. Additional domains, according to Interpro annotations, were searched in 2699 plant NLR proteins from different families of canonical NLRs. The NLR subgroups defined by Greenphyl are shown with different colors: orange (GP015065), red (GP015132), blue (GP015056) and black (TIR‐NB‐LRR: GP000012). (a) The number of unusual domains and the number of NLRs analyzed is indicated above each bar (see Supporting Information Table S1 for more detail). The 1393 canonical receptor‐like kinases (RLKs) from Greenphyl family GP039790 (gray bars) were used as a control to evaluate, in this different set of multi‐domain immune receptor proteins, the frequency of such unusual domains (see Table S2 for more detail). (b) The 22 plant genomes showing the highest frequencies and (c) the number of NLR proteins carrying the most frequent domains (IPR011009, IPR003657 and IPR003656) are shown.

Figure 3

The ZBED protein is involved in blast resistance. The role of the rice ZBED protein containing three BED domains in disease resistance against the blast fungus Magnaporthe oryzae was evaluated by over‐expressing (Supporting Information Fig. S1b–d) and knocking out (Fig. S1a) the ZBED gene. The three homozygous, monolocus T3 transgenic rice lines over‐expressing the ZBED gene (a) showed fewer disease lesions (b, c). By contrast, the insertion mutant for the ZBED gene (d) showed more disease symptoms (e, f). ZBED gene expression was measured by qRT‐PCR (normalized by Actin). Disease lesions, characterized by sporulation and a grayish center, were quantified 7 d after inoculation with the M. oryzae strain GUY11 and representative symptoms are shown (c, f). Such differences in symptom strength between (c) and (f) are frequent in this type of plant–pathogen interaction study and are probably attributable to variability in plant growth conditions. A Student t‐test was used to compare the over‐expresser lines (OX) and the knock‐out (ko) mutant line with their respective null‐segregant controls (NS; see the Materials and Methods section): *, P < 0.05, **, P < 0.01; ***, P < 0.001. The gene expression and disease values are the mean + SD from three independent experiments.