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

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.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

Functional diversification gave rise to allelic specialization in a rice NLR immune receptor pair.

Cooperation between receptors from the nucleotide-binding, leucine-rich repeats (NLR) superfamily is important for intracellular activation of immune responses. NLRs can function in pairs that, upon pathogen recognition, trigger hypersensitive cell death and stop pathogen invasion. Natural selection drives specialization of host immune receptors towards an optimal response, whilst keeping a tight regulation of immunity in the absence of pathogens. However, the molecular basis of co-adaptation and specialization between paired NLRs remains largely unknown. Here, we describe functional specialization in alleles of the rice NLR pair Pik that confers resistance to strains of the blast fungus Magnaporthe oryzae harbouring AVR-Pik effectors. We revealed that matching pairs of allelic Pik NLRs mount effective immune responses, whereas mismatched pairs lead to autoimmune phenotypes, a hallmark of hybrid necrosis in both natural and domesticated plant populations. We further showed that allelic specialization is largely underpinned by a single amino acid polymorphism that determines preferential association between matching pairs of Pik NLRs. These results provide a framework for how functionally linked immune receptors undergo co-adaptation to provide an effective and regulated immune response against pathogens. Understanding the molecular constraints that shape paired NLR evolution has implications beyond plant immunity given that hybrid necrosis can drive reproductive isolation.

Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain.

A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

The Blast Fungus Decoded: Genomes in Flux.

Plant disease outbreaks caused by fungi are a chronic threat to global food security. A prime case is blast disease, which is caused by the ascomycete fungus Magnaporthe oryzae (syn. Pyricularia oryzae), which is infamous as the most destructive disease of the staple crop rice. However, despite its Linnaean binomial name, M. oryzae is a multihost pathogen that infects more than 50 species of grasses. A timely study by P. Gladieux and colleagues (mBio 9:e01219-17, 2018, https://doi.org/10.1128/mBio.01219-17) reports the most extensive population genomic analysis of the blast fungus thus far. M. oryzae consists of an assemblage of differentiated lineages that tend to be associated with particular host genera. Nonetheless, there is clear evidence of gene flow between lineages consistent with maintaining M. oryzae as a single species. Here, we discuss these findings with an emphasis on the ecologic and genetic mechanisms underpinning gene flow. This work also bears practical implications for diagnostics, surveillance, and management of blast diseases.

The coming of age of EvoMPMI: evolutionary molecular plant-microbe interactions across multiple timescales.

Plant-microbe interactions are great model systems to study co-evolutionary dynamics across multiple timescales. However, mechanistic research on plant-microbe interactions has often been conducted with little consideration of evolutionary concepts and methods. Conversely, evolutionary research has rarely integrated the range of mechanisms and models from the molecular plant-microbe interactions field. In recent years, the incipient field of evolutionary molecular plant-microbe interactions (EvoMPMI) has emerged to bridge this gap. Here, we report on some of the recent advances in EvoMPMI. In particular, we highlight new systems to study microbe interactions with early diverging land plants, and new findings from studies of adaptive evolution in pathogens and plants. By linking mechanistic and evolutionary research, EvoMPMI promises to expand our understanding of plant-microbe interactions.

Lessons in Effector and NLR Biology of Plant-Microbe Systems.

A diversity of plant-associated organisms secrete effectors-proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat region (NLR)-containing proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.

Nine things to know about elicitins.

888 I. 888 II. 889 III. 889 IV. 889 V. 891 VI. 891 VII. 891 VIII. 892 IX. 892 X. 893 XI. 893 893 References 893 SUMMARY: Elicitins are structurally conserved extracellular proteins in Phytophthora and Pythium oomycete pathogen species. They were first described in the late 1980s as abundant proteins in Phytophthora culture filtrates that have the capacity to elicit hypersensitive (HR) cell death and disease resistance in tobacco. Later, they became well-established as having features of microbe-associated molecular patterns (MAMPs) and to elicit defences in a variety of plant species. Research on elicitins culminated in the recent cloning of the elicitin response (ELR) cell surface receptor-like protein, from the wild potato Solanum microdontum, which mediates response to a broad range of elicitins. In this review, we provide an overview on elicitins and the plant responses they elicit. We summarize the state of the art by describing what we consider to be the nine most important features of elicitin biology.

Homocysteine, folic acid and vitamin B12 concentration in patients with recurrent miscarriages.

OBJECTIVES: The aim of the project was the assessment of clinical usefulness of the determination of blood serum homocysteine concentration, folic acid and vitamin B12 in recurrent miscarriages. METHODS: 30 non-pregnant women with recurrent miscarriages (examined group-I) and for 20 non-pregnant women without obstetric failures in medical history (control group-II) were examined. RESULTS: In the examined group (group I), the average concentration of homocysteine (9,45 micromol/l) was not statistically higher in comparison to the control group (group II) (8,47 micromol/l) (p>0,05). In group I the average vitamin B12 concentration in blood serum was 178,3 pg/ml and it was statistically lower (p<0,001) in comparison with the control group (II) (268,6 pg/ml). Such a relation was not observed for the vitamin B12, where the average concentration of this parameter was not dependent on the miscarriage number. A high negative correlation (R= -0,5397, p<0,01) was observed between the level of folic acid and homocysteine concentration in the group of women with recurrent miscarriages and a very high negative correlation (r = -0,9586 p<0,001) in the control group. No relation (R=0,0992 p>0,05) between the average concentration of vitamin B12 in blood serum and the average homocysteine in the nullipara group with recurrent miscarriages CONCLUSIONS: Together with the increasing number of abortions, the average homocysteine concentrations grew and the average folic acid concentrations lowered.

Homocysteine serum concentration and uterine artery color Doppler examination in cases of recurrent miscarriages with unexplained etiology.

OBJECTIVES: To study the assessment of diagnostic value and clinical usefulness of the determination of homocysteine concentration in blood serum in cases of recurrent miscarriages and the relation between the concentration of homocysteine in blood serum and parameter values determining the Doppler blood flow in the uterine arteries. METHODS: Homocysteine concentration in blood serum was determined in a group of 30 women with at least two subsequent miscarriages with no clear reason and in the control group consisted of 20 non-pregnant women without a medical history of obstetric failures, having at least one healthy child. In all cases Color Doppler sonography was performed to determine flow velocity waveforms of the uterine arteries in luteal phase of the menstrual cycle. RESULTS: Both pulsatility (PI) and resistance indices (RI) were considerably higher (p<0.01, p<0.05) for the group of women with recurrent abortions. In the group of women with obstetrical failures high positive correlation (R=0.6903, p<0.001) and in the control group very high positive correlation (r=0.8163, p<0.001) was found, between average values of PI and average HC concentration. High positive correlation (R=0.6260, p<0.001) in the examined group and very high positive correlation (r=0.9201, p<0.001) in the control group was obtained between average values of RI, and average HC concentrations in blood serum . CONCLUSIONS: The recurring miscarriages occur in connection with the elevated homocysteine concentration, in consequence they can point out the pathology within the uterine-fetal blood vessels.