Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)‐dependent resistance and for activation of jasmonate (JA)‐dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two‐hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome‐wide transcript profiling indicated that MKS1 is required for full SA‐dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild‐type plants is sufficient to activate SA‐dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two‐hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA‐related defense gene PR1 . MKS1 may therefore contribute to MPK4‐regulated defense activation by coupling the kinase to specific WRKY transcription factors.

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3- to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 andCYP79B3 were both highly induced. Specifically, the indole glucosinolateN-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutantcoi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducingmpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.

In order to get global molecular understanding of one of the most important crop diseases worldwide, we investigated compatible and incompatible interactions between Phytophthora infestans and potato (Solanum tuberosum). We used the two most field-resistant potato clones under Swedish growing conditions, which have the greatest known local diversity of P. infestans populations, and a reference compatible cultivar. Quantitative label-free proteomics of 51 apoplastic secretome samples (PXD000435) in combination with genome-wide transcript analysis by 42 microarrays (E-MTAB-1515) were used to capture changes in protein abundance and gene expression at 6, 24 and 72 hours after inoculation with P. infestans. To aid mass spectrometry analysis we generated cultivar-specific RNA-seq data (E-MTAB-1712), which increased peptide identifications by 17%. Components induced only during incompatible interactions, which are candidates for hypersensitive response initiation, include a Kunitz-like protease inhibitor, transcription factors and an RCR3-like protein. More secreted proteins had lower abundance in the compatible interaction compared to the incompatible interactions. Based on this observation and because the well-characterized effector-target C14 protease follows this pattern, we suggest 40 putative effector targets. In summary, over 17000 transcripts and 1000 secreted proteins changed in abundance in at least one time point, illustrating the dynamics of plant responses to a hemibiotroph. Half of the differentially abundant proteins showed a corresponding change at the transcript level. Many putative hypersensitive and effector-target proteins were single representatives of large gene families.