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Study on the regulation of broad-spectrum resistance is an active area in plant biology. RESISTANCE TO POWDERY MILDEW 8.1 (RPW8.1) is one of a few broad-spectrum resistance genes triggering the hypersensitive response (HR) to restrict multiple pathogenic infections. To address the question how RPW8.1 signaling is regulated, we performed a genetic screen and tried to identify mutations enhancing RPW8.1-mediated HR. Here, we provided evidence to connect an annexin protein with RPW8.1-mediated resistance in Arabidopsis against powdery mildew. We isolated and characterized Arabidopsis b7-6 mutant. A point mutation in b7-6 at the At5g12380 locus resulted in an amino acid substitution in ANNEXIN 8 (AtANN8). Loss-of-function or RNA-silencing of AtANN8 led to enhanced expression of RPW8.1, RPW8.1-dependent necrotic lesions in leaves, and defense against powdery mildew. Conversely, over-expression of AtANN8 compromised RPW8.1-mediated disease resistance and cell death. Interestingly, the mutation in AtANN8 enhanced RPW8.1-triggered H 2 O 2 . In addition, mutation in AtANN8 led to hypersensitivity to salt stress. Together, our data indicate that AtANN8 is involved in multiple stress signaling pathways and negatively regulates RPW8.1-mediated resistance against powdery mildew and cell death, thus linking ANNEXIN's function with plant immunity., (© 2020 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

miRNAs contribute to plant resistance against pathogens. Previously, we found that the function of miR398b in immunity in rice differs from that in Arabidopsis. However, the underlying mechanisms are unclear. In this study, we characterized the mutants of miR398b target genes and demonstrated that multiple superoxide dismutase genes contribute to miR398b-regulated rice immunity against the blast fungus Magnaporthe oryzae. Out of the four target genes of miR398b, mutations in Cu/Zn-Superoxidase Dismutase1 (CSD1), CSD2 and Os11g09780 (Superoxide DismutaseX, SODX) led to enhanced resistance to M. oryzae and increased hydrogen peroxide (H 2 O 2 ) accumulation. By contrast, mutations in Copper Chaperone for Superoxide Dismutase (CCSD) resulted in enhanced susceptibility. Biochemical studies revealed that csd1, csd2 and sodx displayed altered expression of CSDs and other superoxide dismutase (SOD) family members, leading to increased total SOD enzyme activity that positively contributed to higher H 2 O 2 production. By contrast, the ccsd mutant showed CSD protein deletion, resulting in decreased CSD and total SOD enzyme activity. Our results demonstrate the roles of different SODs in miR398b-regulated resistance to rice blast disease, and uncover an integrative regulatory network in which miR398b boosts total SOD activity to upregulate H 2 O 2 concentration and thereby improve disease resistance., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The extrahaustorial membrane (EHM) is a host-derived interfacial membrane encasing the haustorium of powdery mildew fungi. Arabidopsis thaliana RESISTANCE TO POWDERY MILDEW 8.2 (RPW8.2) is specifically targeted to the EHM via two EHM-targeting signals. Here, we demonstrate that proper coordination between the trafficking forces engaged via the EHM-targeting signals and the nuclear localization signals (NLSs), as well as the nuclear export signals (NESs), in RPW8.2 is critical for the activation of cell death and defense. We show that in the absence of pathogens, RPW8.2 is partitioned between the cytoplasm and the nucleus, and turned over via both the 26S proteasome- and the vacuole-dependent pathways. Enhanced cytoplasmic localization of RPW8.2 by tagging it with a NES led to lethal cell death. By contrast, enhanced nuclear localization of RPW8.2 by adding an NLS to it resulted in resistance to powdery mildew. Whereas expression of the NES-containing C-terminal domain of RPW8.2 in the cytoplasm is sufficient to trigger cell death, no such cell death-inducing activity is found with RPW8.2 variants that contain the two EHM-targeting signals along with the NES-containing C-terminal domain. In addition, we present evidence for the involvement of a leaf senescence pathway in RPW8.2-mediated cell death and defense. Taken together, our data suggest that RPW8.2 is subject to adjustment by distinct and perhaps coordinated mechanisms for its localization and function via interaction with the multiple intramolecular trafficking signals, which should provide further insights into RPW8.2-activated, EHM-focused resistance against powdery mildew., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Broad-spectrum disease resistance is a highly valuable trait in plant breeding and attracts special attention in research. The Arabidopsis gene locus RESISTANCE TO POWDERY MILDEW 8 (RPW8) contains two adjacent homologous genes, RPW8.1 and RPW8.2, and confers broad-spectrum resistance to powdery mildew. Remarkably, the RPW8.2 protein is specifically localized to the extrahaustorial membrane (EHM) encasing the feeding structure of powdery mildew whereby RPW8.2 activates haustorium-targeted defenses. Here, we show that ectopic expression of the yellow fluorescent protein (YFP)-tagged RPW8.1 from the native promoter leads to unique cell death lesions and enhances resistance to virulent fungal and oomycete pathogens that cause powdery mildew and downy mildew diseases, respectively. In powdery mildew-infected plants, RPW8.1-YFP accumulates at higher levels in the mesophyll cells underneath the infected epidermal cells where RPW8.2-YFP is mainly expressed. This cell type-preferential protein accumulation pattern largely correlates with that of H(2)O(2) accumulation, suggesting that RPW8.1 may spatially collaborate with RPW8.2 in activation of resistance to powdery mildew. Interestingly, when ectopically expressed from the RPW8.2 promoter, RPW8.1-YFP is also targeted to the EHM of powdery mildew and the transgenic plants display resistance to both powdery mildew and downy mildew. Using YFP as a reporter, we further reveal that the RPW8.1 promoter is constitutively active but induced to higher levels in cells at the infection site, whereas the RPW8.2 promoter is activated specifically in cells at the infection site. Taken together, our results suggest that RPW8.1 (and its promoter) is functionally distinct from RPW8.2 and may have a higher potential in engineering broad-spectrum resistance in plants., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fitness cost is a common phenomenon in rice blast disease-resistance breeding. MiR396 is a highly conserved microRNA (miRNA) family targeting Growth Regulating Factor (OsGRF) genes. Mutation at the target site of miR396 in certain OsGRF gene or blocking miR396 expression leads to increased grain yield. Here we demonstrated that fitness cost can be trade-off in miR396- OsGRF s module via balancing growth and immunity against the blast fungus. The accumulation of miR396 isoforms was significantly increased in a susceptible accession, but fluctuated in a resistant accession upon infection of Magnaporthe oryzae. The transgenic lines over-expressing different miR396 isoforms were highly susceptible to M. oryzae. In contrast, overexpressing target mimicry of miR396 to block its function led to enhanced resistance to M. oryzae in addition to improved yield traits. Moreover, transgenic plants overexpressing OsGRF6 , OsGRF7 , OsGRF8 , and OsGRF9 exhibited enhanced resistance to M. oryzae , but showed different alteration of growth. While overexpression of OsGRF7 led to defects in growth, overexpression of OsGRF6 , OsGRF8 , and OsGRF9 resulted in better or no significant change of yield traits. Collectively, our results indicate that miR396 negatively regulates rice blast disease- resistance via suppressing multiple OsGRF s, which in turn differentially control growth and yield. Therefore, miR396- OsGRFs could be a potential module to demolish fitness cost in rice blast disease-resistance breeding. [ABSTRACT FROM AUTHOR]

Summary: The Arabidopsis gene RESISTANCE TO POWDERY MILDEW8.1 (RPW8.1) confers resistance to virulent fungal and oomycete pathogens that cause powdery mildew and downy mildew, respectively. However, the underlying mechanism remains unclear. Here, we show that ectopic expression of RPW8.1 boosts pattern‐triggered immunity (PTI) resulting in enhanced resistance against different pathogens in both Arabidopsis and rice. In Arabidopsis, transcriptome analysis revealed that ectopic expression of RPW8.1‐YFP constitutively up‐regulates expression of many pathogen‐associated molecular pattern (PAMP‐)‐inducible genes. Consistently, upon PAMP application, the transgenic line expressing RPW8.1‐YFP exhibited more pronounced PTI responses such as callose deposition, production of reactive oxygen species, expression of defence‐related genes and hypersensitive response‐like cell death. Accordingly, the growth of a virulent bacterial pathogen was significantly inhibited in the transgenic lines expressing RPW8.1‐YFP. Conversely, impairment of the PTI signalling pathway from PAMP cognition to the immediate downstream relay of phosphorylation abolished or significantly compromised RPW8.1‐boosted PTI responses. In rice, heterologous expression of RPW8.1‐YFP also led to enhanced resistance to the blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Taken together, our data suggest a surprising mechanistic connection between RPW8.1 function and PTI, and demonstrate the potential of RPW8.1 as a transgene for engineering disease resistance across wide taxonomic lineages of plants. [ABSTRACT FROM AUTHOR]

The ectopic expression of Arabidopsis gene RESISTANCE TO POWDERY MILDEW 8.1 (R1Y4 plant line) triggers a hypersensitive response (HR) providing defense potentially effective against different pathogens. In order to understand the regulation of RPW8.1- mediated signaling, an approach based on ethyl methanesulfonate (EMS) mutagenesis was adopted to identify mutants that enhance or impair RPW8.1 -mediated cell death. Here we describe the identification of mutants b10-6 and b9-1, point mutations that were induced in the At2g37630 locus resulting in an amino acid substitution or a premature stop codon in ASYMMETRIC LEAVES 1 (AS1), respectively. Loss-of-function in AS1 altered the transcription of RPW8.1 , RPW8.1 -dependent necrotic lesions in leaves and resistance to powdery mildew. In the mutant b10-6, RPW8.1 -mediated cell death and disease resistance was compromised, however. in b9-1 RPW8.1 -mediated cell death and resistance to powdery mildew was slightly enhanced. Expression of AS1-eYFP within b10-6 background suppressed RPW8.1 -mediated cell death and disease resistance in the short-day condition, but largely restored the phenotypes of b10-6 to that of R1Y4 in the long-day condition. These data imply that proper expression of AS1 is required for full function of RPW8.1 in triggering cell death and resistance to powdery mildew. [ABSTRACT FROM AUTHOR]

Arabidopsis RESISTANCE TO POWDERY MILDEW 8.1(RPW8.1) confers broad-spectrum resistance to both fungal and bacterial pathogens. To uncover the regulatory mechanism of RPW8.1 -mediated resistance, we constructed an EMS mutagenesis library based on the Arabidopsis transgenic line R1Y4 expressing RPW8.1-YFP from native promoter. Here, we described a variegation mutant B26m1-4 from the library. Map-based cloning identified a G-to-A point mutation at the border between the 7th intron and the 8th exon of IMMUTANS (IM , AT4G22260) locus, leading to deletion of the entire 8th exon. Although the variegation phenotype is independent of RPW8.1 , mutation in IM significantly compromised RPW8.1 -mediated resistance against powdery mildew and bacterial pathogens, which were associated with attenuated defense responses, such as H 2 O 2 accumulation, cell death, and callose deposition. Taken together, these data indicate that IM positively regulates RPW8.1 -mediated disease resistance. • We identified a variegation mutant and cloned its responsible gene as IM. • IM is required for full function of RPW8.1in resistance to fungal and bacterial pathogens. • IM mutation attenuates defense responses in RPW8.1-expressing Arabidopsis. • The variegation phenotype of im is independent of RPW8.1. [ABSTRACT FROM AUTHOR]