Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved effector proteins to counteract antimicrobial autophagy. How adapted pathogens co-opt autophagy for their own benefit is poorly understood. The Irish famine pathogen Phytophthora infestans secretes the effector protein PexRD54 that selectively activates an unknown plant autophagy pathway that antagonizes antimicrobial autophagy at the pathogen interface. Here, we show that PexRD54 induces autophagosome formation by bridging vesicles decorated by the small GTPase Rab8a with autophagic compartments labeled by the core autophagy protein ATG8CL. Rab8a is required for pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing specific trafficking pathways underpin selective autophagy. By subverting Rab8a-mediated vesicle trafficking, PexRD54 utilizes lipid droplets to facilitate biogenesis of autophagosomes diverted to pathogen feeding sites. Altogether, we show that PexRD54 mimics starvation-induced autophagy to subvert endomembrane trafficking at the host-pathogen interface, revealing how effectors bridge distinct host compartments to expedite colonization.

Plants are continuously challenged by pathogens, affecting most staple crops compromising food security. They have evolved different mechanisms to counterattack pathogen infection, including the accumulation of pathogenesis-related (PR) proteins. These proteins have been implicated in active defense, and their overexpression has led to enhanced resistance in nuclear transgenic plants, although in many cases constitutive expression resulted in lesion-mimic phenotypes. We decided to evaluate plastid transformation as an alternative to overcome limitations observed for nuclear transgenic technologies. The advantages include the possibilities to express polycistronic RNAs, to obtain higher protein expression levels, and the impeded gene flow due to the maternal inheritance of the plastome. We transformed Nicotiana tabacum plastids to co-express the tobacco PR proteins AP24 and β-1,3-glucanase. Transplastomic tobacco lines were characterized and subsequently challenged with Rhizoctonia solani , Peronospora hyoscyami f.sp. tabacina and Phytophthora nicotianae . Results showed that transplastomic plants expressing AP24 and β-1,3-glucanase are resistant to R . solani in greenhouse conditions and, furthermore, they are protected against P . hyoscyami f.sp. tabacina and P . nicotianae in field conditions under high inoculum pressure. Our results suggest that plastid co- expression of PR proteins AP24 and β-1,3-glucanase resulted in enhanced resistance against filamentous pathogens.

We describe the potato CDPK family and place StCDPK7 as a player in potato response to Phytophthora infestans infection, identifying phenylalanine ammonia lyase as its specific phosphorylation target in vitro. Calcium-dependent protein kinases (CDPKs) decode calcium (Ca) signals and activate different signaling pathways involved in hormone signaling, plant growth, development, and both abiotic and biotic stress responses. In this study, we describe the potato CDPK/CRK multigene family; bioinformatic analysis allowed us to identify 20 new CDPK isoforms, three CDPK-related kinases (CRKs), and a CDPK-like kinase. Phylogenetic analysis indicated that 26 StCDPKs can be classified into four groups, whose members are predicted to undergo different acylation patterns and exhibited diverse expression levels in different tissues and in response to various stimuli. With the aim of characterizing those members that are particularly involved in plant-pathogen interaction, we focused on StCDPK7. Tissue expression profile revealed that StCDPK7 transcript levels are high in swollen stolons, roots, and mini tubers. Moreover, its expression is induced upon Phytophthora infestans infection in systemic leaves. Transient expression assays showed that StCDPK7 displays a cytosolic/nuclear localization in spite of having a predicted chloroplast transit peptide. The recombinant protein, StCDPK7:6xHis, is an active Ca-dependent protein kinase that can phosphorylate phenylalanine ammonia lyase, an enzyme involved in plant defense response. The analysis of the potato CDPK family provides the first step towards the identification of CDPK isoforms involved in biotic stress. StCDPK7 emerges as a relevant player that could be manipulated to deploy disease resistance in potato crops.

Eukaryotic cells deploy autophagy to eliminate invading microbes. In turn, pathogens have evolved 22 effector proteins to counteract antimicrobial autophagy. How and why adapted pathogens co-opt 23 autophagy for their own benefit is poorly understood. The Irish famine pathogen Phythophthora 24 infestans secretes the effector protein PexRD54 that selectively activates an unknown plant 25 autophagy pathway, while antagonizing antimicrobial autophagy. Here we show that PexRD54 26 induces autophagosome formation by bridging small GTPase Rab8a-decorated vesicles with 27 autophagic compartments labelled by the core autophagy protein ATG8CL. Rab8a is required for 28 pathogen-triggered and starvation-induced but not antimicrobial autophagy, revealing that specific 29 trafficking pathways underpin selective autophagy. We discovered that Rab8a contributes to basal 30 immunity against P. infestans, but PexRD54 diverts a sub-population of Rab8a vesicles to lipid 31 droplets that associate with autophagosomes. These are then diverted towards pathogen feeding 32 structures that are accommodated within the host cells. We propose that PexRD54 mimics starvation-33 induced autophagy by channeling host endomembrane trafficking towards the pathogen interface 34 possibly to acquire nutrients. This work reveals that effectors can interconnect independent host 35 compartments to stimulate complex cellular processes that benefit the pathogen. Graphical abstract: 37 38 39 40 41 42 43 44 45 Introduction: 46 47Autophagy is a conserved eukaryotic cellular process that mediates the lysosomal degradation and 48 relocation of cellular cargoes within double-membraned vesicles called autophagosomes (He and 49 Klionsky, 2009; Leidal et al., 2020). Although autophagy is historically considered to be a bulk 50 catabolic pathway tasked with maintaining cellular homeostasis under normal or stress conditions, it 51 is now clear that autophagy can be highly selective in cargo choice (Zaffagnini and Martens, 2016). 52Autophagic cargoes are typically captured during autophagosome formation, a complex process that 53 is regulated by the concerted action of a set of conserved autophagy related proteins (ATG) as well 54 as specialized autophagy adaptors and cargo receptors (Mizushima et al., 2011). These captured 55 cargoes are sorted within the autophagosome during maturation of the isolation membrane (also 56 known as the phagophore) via the specific interactions between cargo receptors and a lipidated form 57 of ATG8, which decorates the phagophore to serve as a docking platform for cargo receptors 58 (Ryabovol and Minibayeva, 2016; Slobodkin and Elazar, 2013). 60The source of the phagophore is still under debate, but its primary source is considered to be the 61 endoplasmic reticulum (ER) (Bernard and Klionsky, 2013; Hamasaki et al., 2013). As the cargo is 62 being captured, the phagophore undergoes massive expansion and finally gets sealed to form a 63 mature autophagosome. Therefore, formation of the autophagosome requires additional lipid supplies 64...