Fungal pathogens deploy a barrage of secreted effectors to subvert host immunity, often by evading, disrupting, or altering key components of transcription, defense signaling, and metabolic pathways. However, the underlying mechanisms of effectors and their host targets are largely unexplored in necrotrophic fungal pathogens. Here, we describe the effector protein Ascochyta rabiei PEXEL-like Effector Candidate 25 (ArPEC25), which is secreted by the necrotroph A. rabiei, the causal agent of Ascochyta blight disease in chickpea (Cicer arietinum), and is indispensable for virulence. After entering host cells, ArPEC25 localizes to the nucleus and targets the host LIM transcription factor CaβLIM1a. CaβLIM1a is a transcriptional regulator of CaPAL1, which encodes phenylalanine ammonia lyase (PAL), the regulatory, gatekeeping enzyme of the phenylpropanoid pathway. ArPEC25 inhibits the transactivation of CaβLIM1a by interfering with its DNA-binding ability, resulting in negative regulation of the phenylpropanoid pathway and decreased levels of intermediates of lignin biosynthesis, thereby suppressing lignin production. Our findings illustrate the role of fungal effectors in enhancing virulence by targeting a key defense pathway that leads to the biosynthesis of various secondary metabolites and antifungal compounds. This study provides a template for the study of less explored necrotrophic effectors and their host target functions.

Polarized hyphal growth of filamentous pathogenic fungi is an essential event for host penetration and colonization. The long-range early endosomal trafficking during hyphal growth is crucial for nutrient uptake, sensing of host-specific cues, and regulation of effector production. Bin1/Amphiphysin/Rvs167 (BAR) domain-containing proteins mediate fundamental cellular processes, including membrane remodeling and endocytosis. Here, we identified a F-BAR domain protein (ArF-BAR) in the necrotrophic fungus Ascochyta rabiei and demonstrate its involvement in endosome-dependent fungal virulence on the host plant Cicer arietinum. We show that ArF-BAR regulates endocytosis at the hyphal tip, localizes to the early endosomes, and is involved in actin dynamics. Functional studies involving gene knockout and complementation experiments reveal that ArF-BAR is necessary for virulence. The loss-of-function of ArF-BAR gene results in delayed formation of apical septum in fungal cells near growing hyphal tip that is crucial for host penetration, and impaired secretion of a candidate effector having secretory signal peptide for translocation across the endoplasmic reticulum membrane. The mRNA transcripts of ArF-BAR were induced in response to oxidative stress and infection. We also show that ArF-BAR is able to tubulate synthetic liposomes, suggesting the functional role of F-BAR domain in membrane tubule formation in vivo. Further, our studies identified a stress-induced transcription factor, ArCRZ1 (Calcineurin-responsive zinc finger 1), as key transcriptional regulator of ArF-BAR expression. We propose a model in which ArCRZ1 functions upstream of ArF-BAR to regulate A. rabiei virulence through a mechanism that involves endocytosis, effector secretion, and actin cytoskeleton regulation.

The impact of global climate change has highlighted the need for a better understanding of how plants respond to multiple simultaneous or sequential stresses, not only to gain fundamental knowledge of how plants integrate signals and mount a coordinated response to stresses but also for applications to improve crop resilience to environmental stresses. In recent years, there has been a stronger emphasis on understanding how plants integrate stresses and the molecular mechanisms underlying the crosstalk between the signaling pathways and transcriptional programs that underpin plant responses to multiple stresses. The combination of flooding (or resulting hypoxic stress) with pathogen infection is particularly relevant due to the frequent co-occurrence of both stresses in nature. This review focuses on (i) experimental approaches and challenges associated with the study of combined and sequential flooding/hypoxia and pathogen infection, (ii) how flooding (or resulting hypoxic stress) influences plant immunity and defense responses to pathogens, and (iii) how flooding contributes to shaping the soil microbiome and is linked to plants’ ability to fight pathogen infection.

The ubiquitin‐dependent Arg/N‐degron pathway relates the stability of a substrate protein to the nature of its N‐terminal amino acid residue or its biochemical modifications, with some N‐terminal residues being recognized by specific E3 ubiquitin ligases, resulting in the ubiquitylation and degradation of the substrate protein. Work in the model plant Arabidopsis thaliana has shown that the Arg/N‐degron pathway is a key regulator of plant responses to hypoxia, which can be either physiological or a stress in the context of waterlogging or submergence. The role of the Arg/N‐degron pathway in hypoxia response is mediated via the oxygen‐dependent degradation of group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors, which act as the master regulators of the hypoxia response program in plants. Analysis of Arabidopsis mutants for different enzymatic components of the Arg/N‐degron pathway has also revealed its roles in the regulation of responses to other abiotic stresses (e.g., salt stress), as well as to pathogens. Although much has been learned from studies in Arabidopsis about the functions of the Arg/N‐degron pathway, very little is known about this pathway in crops, including in Brassica crops such as oilseed rape, cabbage, or turnip. To determine functional similarities and divergence of the Arg/N‐degron pathway between Arabidopsis and Brassica crops, we isolated and characterized the first Arg/N‐degron pathway mutants in Brassica rapa (turnip, pak choi), a diploid Brassica crop closely related to oilseed rape. We focused on two enzymatic components, namely, the arginine‐transferases ( ATE s) and the E3 ubiquitin ligase PROTEOLYSIS6 ( PRT6 ). Our results show both similarities and divergence of function for these Arg/N‐degron pathway components in B. rapa compared to Arabidopsis. Specifically, ATE mutants in B. rapa arrest their development at the seedling stage, which contrasts with the mild phenotypic defects of the equivalent Arabidopsis mutants. Double mutant lines for two of the three PRT6 genes in B. rapa indicated a constitutive activation of hypoxia response genes at the transcriptional level, as shown in the single prt6 mutant in Arabidopsis. However, contrary to Arabidopsis, the B. rapa double mutants were more sensitive to waterlogging and hypoxia and did not show differential response to salt stress or to biotic stress compared to the wild type. The functional divergence identified likely reflects variability in each species in the substrate repertoire and/or in the regulation of pathways or targets downstream of Arg/N‐degron pathway substrates. Such differences could be driven by direct selective pressures at N‐termini (e.g., gain or loss of a destabilizing N‐terminal residue) or by species‐specific proteases that may generate destabilizing neo‐N‐termini after cleavage. These similarities and differences highlight the difficulties in translating research findings from Arabidopsis to crops, even within the same plant family (Brassicaceae), and highlight the need to study pathways in crops.

The ubiquitin-dependent Arg/N-degron pathway relates the stability of a substrate protein to the nature of its N-terminal amino acid residue or its biochemical modifications, with some N-terminal residues being recognized by specific E3 ubiquitin ligases, resulting in the ubiquitylation and degradation of the substrate protein. Work in the model plant Arabidopsis thaliana has shown that the Arg/N-degron pathway is a key regulator of plant responses to hypoxia, which can be either physiological or a stress in the context of waterlogging or submergence. The role of the Arg/N-degron pathway in hypoxia response is mediated via the oxygen-dependent degradation of group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factors, which act as the master regulators of the hypoxia response program in plants. Analysis of Arabidopsis mutants for different enzymatic components of the Arg/N-degron pathway has also revealed its roles in the regulation of responses to other abiotic stresses (e.g. salt stress), as well as to pathogens. Although much has been learned from studies in Arabidopsis about the functions of the Arg/N-degron pathway, very little is known about this pathway in crops, including in Brassica crops such as oilseed rape, cabbage or turnip. To determine functional similarities and divergence of the Arg/N-degron pathway between Arabidopsis and Brassica crops, we isolated and characterized the first Arg/N-degron pathway mutants in Brassica rapa (turnip, pak choi), a diploid Brassica crop closely related to oilseed rape. We focused on two enzymatic components, namely the arginine-transferases ( ATE s) and the E3 ubiquitin ligase PROTEOLYSIS6 ( PRT6 ). Our results show both similarities and divergence of function for these Arg/N-degron pathway components in B. rapa compared to Arabidopsis. Specifically, ATE mutants in B. rapa arrest their development at the seedling stage, which contrasts with the mild phenotypic defects of the equivalent Arabidopsis mutants. Double mutant lines for two of the three PRT6 genes in B. rapa indicated a constitutive activation of hypoxia response genes at the transcriptional level, as shown in the single prt6 mutant in Arabidopsis. However, contrary to Arabidopsis, the B. rapa double mutants were more sensitive to waterlogging and hypoxia, and did not show differential response to salt stress or to biotic stress compared to the wild type. The functional divergence identified likely reflects variability in each species in the substrate repertoire and/or in the regulation of pathways or targets downstream of Arg/N-degron pathway substrates. Such differences could be driven by direct selective pressures at N-termini (e.g. gain or loss of a destabilizing N-terminal residue), or by species-specific proteases that may generate destabilizing neo-N-termini after cleavage. These similarities and differences highlight the difficulties in translating research findings from Arabidopsis to crops, even within the same plant family (Brassicaceae) and highlight the need to study pathways in crops.