ATG1 role in the immunity of tomato against Pseudomonas syringae pv. tomato

Abstract

The bacteria Pseudomonas syringae pv. tomato DC3000 (Pst) is widely recognized as a model to study plant immunity. Plants have evolved a two-layered immunity system to defend themselves from pathogens. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are detected by host´s pattern recognition receptors (PRRs) during pattern-triggered immunity (PTI) activation. This leads to changes in the intracellular calcium concentration, production of ROS, activation of MAPK cascades and transcriptional alterations. However virulent pathogens as Pst are able to bypass PTI through the delivery of effector proteins. Resistant plants recognize some of them activating effector-triggered immunity (ETI) that mainly leads to localized programmed cell death (PCD), limiting pathogen growth. Tomato Pto protein kinase interacts with the effectors AvrPto and AvrPtoB and together with Prf lead to activation of ETI response. Using previously generated RNA-seq data we identified two tomato genes (Solyc10g084930 and Solyc09g011320) whose expression is induced during ETI activation. Sequence and phylogeny analysis allowed us establishing that they encode for proteins belonging to the autophagy-related protein 1 (ATG1) group and that each of them has two orthologs in Nicotiana benthamiana, the species we use to test the role of candidate genes in immunity through virus-induced gene silencing (VIGS). Plants silenced with a construct that targets all 4 N. benthamiana orthologs (NbATG1) showed a delay in the development of PCD due to co-expression of Pto and AvrPto, as compared to non-silenced Ec1 control plants. To confirm this result, we challenged N. benthamiana 35S::pto silenced plants with Pseudomonas syringae pv. tabaci (Pstab) expressing AvrPto or an empty vector (EV). We did not observe an increase in disease symptoms with Pstab-AvrPto strain. However, we found a delay in the development of Pstab-EV disease symptoms. To test whether silencing the 4 N. benthamiana orthologs is required for the observed phenotype, we generated two constructs (6008 and 1011) to target them by pairs. We found a similar overall phenotype trend, but with a more marked delay in symptoms when silencing with 1011 and NbATG1 constructs. It is worth noticing that we have not observed growth or development abnormalities in plants silenced with any of the constructs used. We chose a well established transcriptional marker of autophagy (ATG8a) to investigate if silencing of our candidates affects this process. Using qPCR we did not observe differences in ATG8a transcript level between Ec1- and NbATG1-silenced plants, suggesting that at least in unchallenged plants, autophagy is not affected by knocking-down the genes under study. Based on our results, we believe that this group of autophagy-related proteins would be involved in two different pathways, playing a role during ETI-associated PCD and also in the development of disease symptoms.