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The soybean cyst nematode (SCN; Heterodera glycines) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, the presence of a signal peptide, and the lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots, while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Competing Interests: The author(s) declare no conflict of interest.

The soybean cyst nematode (SCN; Heterodera glycines ) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, the presence of a signal peptide, and the lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots, while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

The redox imbalanced 6 mutant (rimb6) of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR) GENES 1, CPR1 (At4g12560), leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA) accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.

To investigate the tolerance factor of Saccharomyces cerevisiae KNU5377 against various types of environmental stress during fermentation, we identified the protein that is upregulated at high temperatures. The highly upregulated protein was high-score-matched as a cytoplasmic peptidyl-prolyl cis-trans isomerase, cyclophilin (Cpr1p), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). We constructed a CPR1-deleted KNU5377 strain (KNU5377Y cpr1Δ) to determine the roles of the protein under fermentative or stress condition. The growth of the S. cerevisiae KNU5377Y cpr1Δ strain was completely inhibited under the following conditions: heat (40°C), hydrogen peroxide (20–30 mM), menadione (0.3 mM), ethanol (16%), sulfuric acid (5 mm), and lactic acid (0.4–0.8%). However, the wild-type and cpr1Δ mutant of S. cerevisiae BY4741 as a positive control did not show differences in sensitivity to stress. It is interesting to note that the wild-type KNU5377Y and KNU5377Y cpr1Δ mutant showed high sensitivity against various stresses, particularly, acid stress such as in the presence of sulfuric and lactic acid. Although the alcohol fermentation rate of the KNU5377Y cpr1Δ mutant markedly decreased with an increase in temperature up to 40°C, we observed no decrease in that of the wild-type strain under the same conditions. These results suggest that CPR1 contributes to the stress tolerance of KNU5377 against various types of environmental stress caused during fermentation, thus leading to the physiological role of maintaining an alcohol fermentation yield, even at high temperatures such as 40°C. [Copyright &y& Elsevier]

The redox imbalanced 6 mutant ( rimb6) of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR) GENES 1, CPR1 (At4g12560), leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA) accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.