Multiple variants of the blast fungus effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19 with high affinity

Abstract

Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. Non-canonical integrated domains are widespread in plant NLRs and are thought to resemble the host target of the recognised effector. AVR-Pik interacts with specific rice HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.

Design of a new effector recognition specificity in a plant NLR immune receptor by molecular engineering of its integrated decoy domain

“…In the RGA4/RGA5 and Pik1/Pik2 model systems, detailed structure-function analysis deciphered at atomic scale how IDs contribute to the recognition of fungal effector proteins (Maqbool et al, 2015;Ortiz et al, 2017;Guo et al, 2018;De la Concepcion et al, 2018). These studies established that small HMA proteins have been recruited repeatedly and independently to serve as decoy domains that physically bind effectors like molecular traps (Oikawa et al, 2020;Maidment et al, 2021). These breakthroughs pave the way towards rational structure-guided design of effector-binding domains in these NLRs.…”

“…Many plant NLRs harbor one or multiple noncanonical domains integrated into their structure (Cesari et al, 2014;Kroj et al, 2016;Sarris et al, 2016;Stein et al, 2018;Bailey et al, 2018;Van de Weyer et al, 2019). Some of these integrated domains (ID) were shown to be involved in the specific recognition of effectors and thought to be decoy domains derived from proteins targeted by these effectors (Cesari et al, 2013;Maqbool et al, 2015; Le Roux et al, 2015;Sarris et al, 2015;Oikawa et al, 2020;Maidment et al, 2021). NLR-IDs often cluster genetically and function in combination with a second NLR that acts as a signaling executer for the sensor NLR-ID.…”

The blast pathogen effector AVR-Pik binds and stabilizes rice heavy metal-associated (HMA) proteins to co-opt their function in immunity

“…These findings support the view that NLR integrated domains have evolved from the host targets of pathogen effectors and that HMA-containing proteins are a major host target of plant pathogen effectors. In an independent study, Maidment et al (2020) demonstrate that AVR-Pik binds to OsHIPP19 with nanomolar affinity in vitro and show the interaction of the effector with this sHMA is via an interface conserved with the Pik-1 integrated HMA domains providing further evidence that this effector targets host sHMA proteins.…”

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

“…Effector proteins often interact with multiple members of host-target protein families [47,[63][64][65]. In addition, multiple pathogen effectors can target the same host protein family [35,36,66], possibly to ensure effective host infection or as an evolutionary "bet-hedging" strategy to counteract detection by the plant immune system [65]. APikL2 shares the MAX-effector fold and similar HMA-binding interfaces with the well characterized effector AVR-Pik.…”