Novel effector recognition capacity engineered into a paired NLR complex

Abstract

The Arabidopsis RRS1-R Resistance gene confers recognition of the bacterial acetyltransferase PopP2 and another bacterial effector, AvrRps4. The RRS1-S allele recognizes AvrRps4 but not PopP2. RRS1-R/RRS1-S heterozygotes cannot recognize PopP2. RRS1-R and RRS1-S also suppress the constitutive RPS4-dependent autoactivity of RRS1-Rslh1. Phytoplasmas cause important plant diseases, and their effectors can cause degradation of specific host proteins. We tested whether attaching a pathogen effector-dependent degron to RRS1-R, enabling its degradation by phytoplasma effector SAP05, could derepress RRS1-Rslh1 autoactivity, resulting in SAP05-dependent resistance. In transient assays in tobacco, RRS1-R-derived constructs can confer a hypersensitive response (HR) to SAP05. However, phytoplasma infection assays in transgenic Arabidopsis resulted in delayed disease symptoms but not full resistance. We provide a proof-of-concept strategy utilizing the recessiveness of a plant immune receptor gene to engineer recognition of a pathogen effector that promotes degradation of a specific host protein.

The effector recognition by synthetic sensor NLR receptors requires the concerted action of multiple interfaces within and outside the integrated domain

“…Wang et al created a variant of RRS1 by adding a Phytoplasma effector SAP05-dependent degron domain to the C-terminus of RRS1, which elicited the hypersensitive response (HR) in N. tabacum leaves triggered by the effector but failed to confer full resistance in transgenic Arabidopsis to the Phytoplasma infection 19 . By engineering RGA5-HMA in accordance with Pikp1-HMA, Cesari et al .…”

“…N. benthamiana or N. tabacum has been widely adopted as a convenient heterologous system to assay plant immune responses, such as HR. However, recent studies showed that designer NLR receptors that gained the capability to recognize effectors and trigger HR in N. benthamiana or N. tabacum may not always enable complete resistance in host plants 18, 19 . These studies alert us that using the heterologous overexpression system to assess the synthetic NLRs seems insufficient.…”

“…Wang et al created a variant of RRS1 by adding a Phytoplasma effector SAP05dependent degron domain to the C-terminus of RRS1, which elicited the hypersensitive response (HR) in N. tabacum leaves triggered by the effector but failed to confer full resistance in transgenic Arabidopsis to the Phytoplasma infection 19 . By engineering RGA5-HMA in accordance with Pikp1-HMA, Cesari et al generated two designer NLR receptors, which shared the engineered β2β3β4 interface of RGA5-HMA and gained a high binding affinity to the non-corresponding effector AVR-PikD in addition to the corresponding effector AVR-Pia, but were unable to confer specific blast resistance in transgenic rice plants against M. oryzae strains expressing AVR-PikD 18 .…”

“…Further, Wang et al generated an RRS1 variant by introducing the Phytoplasma effector SAP05-dependent degron domain to the C-terminus of RRS1. This synthetic RRS1 receptor can recognize SAP05 but is uable to confer full resistance in transgenic Arabidopsis against the Phytoplasma 19 . These studies raise a key question as to why de novo effector-binding results in different outcomes in NLR activation in the host plants.…”

Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor

“…Using a different strategy, a protein domain targeted for degradation by the phytoplasma effector SAP05 was fused to the C-terminus of the TIR-NLR RRS1-R. RRS1-R represses the immune cell death- triggering activity of a second TIR-NLR, RPS4. While transient co-expression of the engineered RRS1-R, RPS4 and the phytoplasma effector SAP05 led to cell death in N. tabacum , transgenic A. thaliana plants were not resistant to phytoplasma carrying the SAP05 effector (23).…”