Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor
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Evaluation Summary:
Engineering NLR proteins to improve disease resistance in crop plants is a major goal of the field. This study applies knowledge from structural and evolutionary studies of the rice NLR protein Pik-1 and cognate effector protein AVR-Pik to engineering of new disease resistance genes. The authors nicely demonstrate that it is indeed possible to engineer resistance proteins with broad recognition specificity for the rice blast fungus. The work is of interest to colleagues in synthetic biology, protein engineering and plant-pathogen interactions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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- Plant Biology (eLife)
Abstract
A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.
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Evaluation Summary:
Engineering NLR proteins to improve disease resistance in crop plants is a major goal of the field. This study applies knowledge from structural and evolutionary studies of the rice NLR protein Pik-1 and cognate effector protein AVR-Pik to engineering of new disease resistance genes. The authors nicely demonstrate that it is indeed possible to engineer resistance proteins with broad recognition specificity for the rice blast fungus. The work is of interest to colleagues in synthetic biology, protein engineering and plant-pathogen interactions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The rice sensor NLR protein Pik-1 carries a HMA domain to sense fungal AVR proteins. Past studies have shown that it is possible to modify the HMA domain to change new recognition specificity. However, whether this approach can generate broad-spectrum NLRs that function in rice plants remains to be shown. Prior work from the authors have shown that each of the existing Pik-1 alleles only recognizes some, but not all AVR-Pik alleles. Interestingly, they found that a natural rice target protein HIPP19 is capable of binding to all known AVR-Pik proteins. In the current study, the authors tested the idea that AVR-Pik-binding sequence in HIPP19 could be utilized to engineer Pik-1 protein with broader recognition specificity. Strikingly, the engineered Pikp-1OsHIPP19-mbl7 is capable of recognizing AVR-PikD, C, and …
Reviewer #1 (Public Review):
The rice sensor NLR protein Pik-1 carries a HMA domain to sense fungal AVR proteins. Past studies have shown that it is possible to modify the HMA domain to change new recognition specificity. However, whether this approach can generate broad-spectrum NLRs that function in rice plants remains to be shown. Prior work from the authors have shown that each of the existing Pik-1 alleles only recognizes some, but not all AVR-Pik alleles. Interestingly, they found that a natural rice target protein HIPP19 is capable of binding to all known AVR-Pik proteins. In the current study, the authors tested the idea that AVR-Pik-binding sequence in HIPP19 could be utilized to engineer Pik-1 protein with broader recognition specificity. Strikingly, the engineered Pikp-1OsHIPP19-mbl7 is capable of recognizing AVR-PikD, C, and F, whereas the original Pikp-1 is only capable of recognizing Avr-PikD. This is supported by both HR-elicitation and protein-protein interactions in N. benthamiana plants. The authors further used a structure-guided approach to identify specific amino acids responsible for expanded recognition of AVR proteins. To this end, they show that the Pikp-1SNK-EKE variant is capable of recognizing all three of the aforementioned AVR-Pik proteins. The proper interactions of the newly introduced amino acids with the Avr-Pik proteins were nicely demonstrated with structural work. Most excitingly, the Pikp-1OsHIPP19-mbl7 and Pikp-1SNK-EKE constructs were introduced in to rice plants lacking Pik-1 as stable transgenes. These lines displayed disease resistance to rice blast strains carrying any of the three AVR-Pik proteins. Overall, the study is well executed and shows how knowledge of structural and evolutionary studies can help engineering disease resistance in a major crop plant. The weakness is with the use of a strong promoter to drive the expression of the engineered Pikp-1 variants in rice and a lack of assessment of potential effects on traits.
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Reviewer #2 (Public Review):
The paired rice Pik-1 and Pik-2 NLRs recognize M. oryzae strains carrying AVR-Pik to activate plant defense. To date, five Pik alleles and six AVR-Pik variants have been identified. Pikp-1 is able to recognize the AVR-PikD. In a previous study, Pikp-1NK-KE mutant is engineered and designed to recognize the AVR-PikA and AVR-PikE. However, no naturally occurring or engineered Pik allele is available to respond to AVR-PikC and AVR-PikF. In this study the authors report other new strategies to extend the disease resistance spectrum of Pikp-1. The authors utilize a host target OsHIPP19 with a HMA domain, which could bind all AVR-Pik variants with high affinity, to design a Pikp-1OsHIPP19 chimera that was able to respond to AVR-PikC and AVR-PikF. Meanwhile, the authors mutagenized Pikp-HMA in the guide of the …
Reviewer #2 (Public Review):
The paired rice Pik-1 and Pik-2 NLRs recognize M. oryzae strains carrying AVR-Pik to activate plant defense. To date, five Pik alleles and six AVR-Pik variants have been identified. Pikp-1 is able to recognize the AVR-PikD. In a previous study, Pikp-1NK-KE mutant is engineered and designed to recognize the AVR-PikA and AVR-PikE. However, no naturally occurring or engineered Pik allele is available to respond to AVR-PikC and AVR-PikF. In this study the authors report other new strategies to extend the disease resistance spectrum of Pikp-1. The authors utilize a host target OsHIPP19 with a HMA domain, which could bind all AVR-Pik variants with high affinity, to design a Pikp-1OsHIPP19 chimera that was able to respond to AVR-PikC and AVR-PikF. Meanwhile, the authors mutagenized Pikp-HMA in the guide of the structure of OsHIPP19-HMA and designed a Pikp-1SNK-EKE mutant that was also able to recognize AVR-PikC and AVR-PikF. Importantly, the transgenic rice plants expressing the engineered Pikp-1 variants conferred effective resistance to blast fungus isolates carrying AVR-PikC or AVR-PikF.
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