Structural Basis of Host Autophagy-related Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54

Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease

Cell entry of a host targeting protein of oomycetes requires gp96

This work is supported by the [European Community’s] Seventh Framework Programme [FP7/2007–2013] under grant agreement no. [238550] (L.L., J.D.-U., C.J.S., P.v.W.); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (F.T., I.d.B., C.J.S., S.W., P.v.W.); Newton Global Partnership Award [BB/N005058/1] (F.T., P.v.W.), the University of Aberdeen (A.D.T., T.R., C.J.S., P.v.W.) and Deutsche Forschungsgemeinschaft [CRC1093] (P.B., T.S.). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies.Peer reviewedPublisher PD

Computational Prediction and Molecular Characterization of an Oomycete Effector and the Cognate Arabidopsis Resistance Gene

Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph oomycete pathogen of the model plant Arabidopsis thaliana and contains a large set of effector proteins that are translocated to the host to exert virulence functions or trigger immune responses. These effectors are characterized by conserved amino-terminal translocation sequences and highly divergent carboxyl-terminal functional domains. The availability of the Hpa genome sequence allowed the computational prediction of effectors and the development of effector delivery systems enabled validation of the predicted effectors in Arabidopsis. In this study, we identified a novel effector ATR39-1 by computational methods, which was found to trigger a resistance response in the Arabidopsis ecotype Weiningen (Wei-0). The allelic variant of this effector, ATR39-2, is not recognized, and two amino acid residues were identified and shown to be critical for this loss of recognition. The resistance protein responsible for recognition of the ATR39-1 effector in Arabidopsis is RPP39 and was identified by map-based cloning. RPP39 is a member of the CC-NBS-LRR family of resistance proteins and requires the signaling gene NDR1 for full activity. Recognition of ATR39-1 in Wei-0 does not inhibit growth of Hpa strains expressing the effector, suggesting complex mechanisms of pathogen evasion of recognition, and is similar to what has been shown in several other cases of plant-oomycete interactions. Identification of this resistance gene/effector pair adds to our knowledge of plant resistance mechanisms and provides the basis for further functional analyses

Structural Elucidation and Functional Characterization of the Hyaloperonospora arabidopsidis Effector Protein ATR13

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew on the model plant Arabidopsis thaliana and has been adapted as a model system to investigate pathogen virulence strategies and plant disease resistance mechanisms. Recognition of Hpa infection occurs when plant resistance proteins (R-genes) detect the presence or activity of pathogen-derived protein effectors delivered to the plant host. This study examines the Hpa effector ATR13 Emco5 and its recognition by RPP13-Nd, the cognate R-gene that triggers programmed cell death (HR) in the presence of recognized ATR13 variants. Herein, we use NMR to solve the backbone structure of ATR13 Emco5, revealing both a helical domain and a disordered internal loop. Additionally, we use site-directed and random mutagenesis to identify several amino acid residues involved in the recognition response conferred by RPP13-Nd. Using our structure as a scaffold, we map these residues to one of two surface-exposed patches of residues under diversifying selection. Exploring possible roles of the disordered region within the ATR13 structure, we perform domain swapping experiments and identify a peptide sequence involved in nucleolar localization. We conclude that ATR13 is a highly dynamic protein with no clear structural homologues that contains two surface-exposed patches of polymorphism, only one of which is involved in RPP13-Nd recognition specificity

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co‐immunoprecipitation (Co‐IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co‐IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co‐localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH‐sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR‐1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase‐mediated vesicular secretion of antimicrobial PR‐1, PDF1.2 and possibly other defence‐related compounds

BAH1/NLA, a RING-Type Ubiquitin E3 Ligase, Regulates the Accumulation of Salicylic Acid and Immune Responses to Pseudomonas syringae DC30001[W][OA]

Salicylic acid (SA) is a primary factor responsible for exerting diverse immune responses in plants and is synthesized in response to attack by a wide range of pathogens. The Arabidopsis (Arabidopsis thaliana) sid2 mutant is defective in a SA biosynthetic pathway involving ISOCHORISMATE SYNTHASE1 (ICS1) and consequently contains reduced levels of SA. However, the sid2 mutant as well as ICS-suppressed tobacco (Nicotiana benthamiana) still accumulate a small but significant level of SA. These observations along with previous studies suggest that SA might also be synthesized by another pathway involving benzoic acid (BA). Here we isolated a benzoic acid hypersensitive1-Dominant (bah1-D) mutant that excessively accumulated SA after application of BA from activation-tagged lines. This mutant also accumulated higher levels of SA after inoculation with Pseudomonas syringae pv tomato DC3000. Analysis of the bah1-D sid2 double mutant suggested that the bah1-D mutation caused both ICS1-dependent and -independent accumulation. In addition, the bah1-D mutant showed SA-dependent localized cell death in response to P. syringae pv tomato DC3000. The T-DNA insertional mutation that caused the bah1-D phenotypes resulted in the suppression of expression of the NLA gene, which encodes a RING-type ubiquitin E3 ligase. These results suggest that BAH1/NLA plays crucial roles in the ubiquitination-mediated regulation of immune responses, including BA- and pathogen-induced SA accumulation, and control of cell death

Knockout of Tobacco Homologs of <i>Arabidopsis Multi-Antibiotic Resistance 1</i> Gene Confers a Limited Resistance to Aminoglycoside Antibiotics

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs–knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants’ sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs

Nicotinamide Effectively Suppresses Fusarium Head Blight in Wheat Plants

Pyridine nucleotides such as a nicotinamide adenine dinucleotide (NAD) are known as plant defense activators. We previously reported that nicotinamide mononucleotide (NMN) enhanced disease resistance against fungal pathogen Fusarium graminearum in barley and Arabidopsis. In this study, we reveal that the pretreatment of nicotinamide (NIM), which does not contain nucleotides, effectively suppresses disease development of Fusarium Head Blight (FHB) in wheat plants. Correspondingly, deoxynivalenol (DON) mycotoxin accumulation was also significantly decreased by NIM pretreatment. A metabolome analysis showed that several antioxidant and antifungal compounds such as trigonelline were significantly accumulated in the NIM-pretreated spikes after inoculation of F. graminearum. In addition, some metabolites involved in the DNA hypomethylation were accumulated in the NIM-pretreated spikes. On the other hand, fungal metabolites DON and ergosterol peroxide were significantly reduced by the NIM pretreatment. Since NIM is relative stable and inexpensive compared with NMN and NAD, it may be more useful for the control of symptoms of FHB and DON accumulation in wheat and other crops