Insights into potato plant immunity reveals Parakletos as a novel ROS suppressor

Potato (Solanum tuberosum L.) is the world's third most commonly grown food crop with very high yield potential. However, its production is hampered by several pathogens, with consequent yield losses. Existing control methods include frequent, costly fungicide applications, and classical breeding is complicated in potato. One way to develop new longterm solutions is to improve understanding of plant molecular immunity and factors capable of providing broad-spectrum resistance. To assist such efforts, comparative proteomic techniques were applied to enhance understanding of changes in protein abundance during the immune responses of potato. These focused on a PTI (Pattern-triggered immunity) model and two ETI (Effector-triggered immunity) models (both related to resistance genes to Phytophthora infestans, the causal agent of late blight) in potato to enhance understanding of changes in protein abundance during its immune responses. Numerous proteins increased in abundance in all observed immune responses, including one identified as sterol transporter protein 2, which is intriguing because sterol content plays an important role in plant immunity, and oomycete pathogens like P. infestans rely on their hosts for sterols. The abundance of RNA binding proteins also changed in different immune reactions. A few proteins changed in abundance in only one of the ETI models, e.g., histones were downregulated in one (ETI responses to P. infestans effector Avr2), whereas a putative multiprotein bridging factor was upregulated in the other (ETI responses to P. infestans effector IpiO). Intriguingly, the proteomic differences between the two ETI models were of similar magnitude to those between the ETI models and PTI. The next step was to study the general PTI-related defence potato proteome by two-step fractionation and new bioinformatics analyses. Five candidates with potential importance in our proteomic dataset were selected for functional validation studies. To facilitate validation studies, a luminol-based assay was developed to study biphasic reactive oxygen species (ROS) bursts in potato leaves induced by flg22 and P. infestans as well as an easy, cost-effective method using near-IR scanning to quantify leaf wounding and disease lesion areas without damaging leaves. Over-expression and silencing of one identified protein, named Parakletos, respectively increased and reduced P. infestans infection in Nicotiana benthamiana. Moreover, its overexpression suppressed the ROS burst response to flg22, while its silencing increased it. Transcript analyses showed upregulation of defence-related genes (e.g., ICS1, PR1, PTI5, and RBOHB) in response to flg22 in Parakletos-silenced plants. Expression of light-harvesting complex B6 (LHCB6) was also enhanced in plants overexpressing Parakletos. It was found that Parakletos co-localized with the Calcium Sensing Receptor (CAS) in chloroplasts, and that it is functionally dependent on CAS. Furthermore, CRISPR/Cas9-mediated knock-out (KO) of parakletos in potato enhanced broad-spectrum resistance to late and early blight in controlled condition. It also reduced infection to Pseudomonas syringae in N. benthamiana. Moreover, during field trials the parakletos-KO lines showed enhanced resistance to P. infestans. These findings contribute to our understanding of plant immunity and provide a new susceptibility-gene based strategy for building broad-spectrum disease resistance in crops