Downy mildew effector recognition and host protein interaction in lettuce

Abstract

In modern agriculture, cultivated lettuce (Lactuca sativa) is typically grown in large monocultures with multiple crops per year and is susceptible to disease outbreaks. In particular the plant pathogenic oomycete Bremia lactucae that causes downy mildew disease on lettuce, has devastating effects and is considered the most important disease affecting the production of cultivated lettuce worldwide. Consequently, the introgression of resistance (R) genes that provide high levels of resistance, is a major aspect of breeding programs. Deployment of R genes exerts strong selection pressure on the pathogen resulting in the rapid breakdown of resistance. Achieving durable resistance against B. lactucae is therefore still a major challenge. Activation of R proteins is dependent on the recognition of effectors. Effector proteins are produced by plant pathogens to facilitate infection. Following the recognition of effectors, an immune response ensues, which is frequently associated with localized cell death (necrosis). In this thesis, we exploited this phenomenon to discover novel R genes. Expression of effector BLR38 resulted in necrosis in the prickly lettuce (Lactuca serriola) accession LS102. Plants that recognized BLR38 were resistant to multiple B. lactucae isolates but not isolate Bl:24 from which BLR38 was cloned. Possibly, isolate Bl:24 contains one or more effectors that are absent in other B. lactucae isolates and effectively interfere with BLR38-triggered immune responses in lettuce. Interestingly, recognition of BLR38 required two loci on distinct chromosomes and was gene dosage dependent. An improved understanding of effector targets in lettuce can help to elucidate the underlying molecular mechanisms leading to disease susceptibility and, potentially, lead to alternative sources of durable resistance. To identify effector targets in lettuce, we performed a protein-protein interaction study using the yeast-two-hybrid (Y2H) system. We identified 61 interactions between 21 effectors and 46 lettuce proteins. Furthermore, the subcellular localization of selected effectors and their plant targets was investigated using confocal microscopy. In nine out of twelve tested combinations the effector and plant target (partly) localized to the same subcellular compartment. Furthermore, we observed in four cases that the effector or the plant target relocalized to the nucleus upon co-expression of their Y2H-identified interaction partner that already resided in the nucleus. These findings strongly suggest that our Y2H screens resulted in the identification of multiple relevant effector targets. The Y2H screens also uncovered the lettuce membrane-associated NAC transcription factor LsNAC069 as target of multiple B. lactucae effectors. LsNAC069 and LsNAC069ΔNAC (a truncated form lacking the NAC domain) localized to the endoplasmic reticulum membrane. LsNAC069ΔNAC also colocalized with effectors BLR05 and BLR09 at the endoplasmic reticulum. We were able to trigger relocalization of LsNAC069ΔNAC to the nucleus by exposing leaves to Phytophthora capsici culture filtrate. Relocalization of LsNAC069ΔNAC was strongly reduced in the presence of the protease inhibitor TPCK indicating that proteolytic cleavage is required for membrane release of LsNAC069ΔNAC. Furthermore, relocalization of LsNAC069ΔNAC was reduced upon co-expression of B. lactucae effectors BLR05, BLR08 and BLR09.