Protein–protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum f. sp. lycopersici resistance protein of the CC-NB-LRR family, were identified. Sequence analysis revealed that I2I-1 belongs to the Formin gene family (SlFormin) whereas I2I-2 has homology to translin-associated protein X (SlTrax). SlFormin required only the N-terminal CC I-2 domain for binding, whereas SlTrax required both I-2 CC and part of the NB-ARC domain. Tomato plants stably silenced for these interactors were not compromised in I-2-mediated disease resistance. When extended or mutated forms of I-2 were used as baits, distinct and often opposite, interaction patterns with the two interactors were observed. These interaction patterns correlated with the proposed activation state of I-2 implying that active and inactive R proteins adopt distinct conformations. It is concluded that the yeast two hybrid system can be used as a proxy to monitor these different conformational states

Potato late blight field resistance from QTL dPI09c is conferred by the NB-LRR gene R8

Following the often short-lived protection that major nucleotide binding, leucine-rich-repeat (NB-LRR) resistance genes offer against the potato pathogen Phytophthora infestans, field resistance was thought to provide a more durable alternative to prevent late blight disease. We previously identified the QTL dPI09c on potato chromosome 9 as a more durable field resistance source against late blight. Here, the resistance QTL was fine-mapped to a 186 kb region. The interval corresponds to a larger, 389 kb, genomic region in the potato reference genome of Solanum tuberosum Group Phureja doubled monoploid clone DM1-3 (DM) and from which functional NB-LRRs R8, R9a, Rpi-moc1, and Rpi-vnt1 have arisen independently in wild species. dRenSeq analysis of parental clones alongside resistant and susceptible bulks of the segregating population B3C1HP showed full sequence representation of R8. This was independently validated using long-range PCR and screening of a bespoke bacterial artificial chromosome library. The latter enabled a comparative analysis of the sequence variation in this locus in diverse Solanaceae. We reveal for the first time that broad spectrum and durable field resistance against P. infestans is conferred by the NB-LRR gene R8, which is thought to provide narrow spectrum race-specific resistance

Nucleotide-binding and molecular interactions of plant disease resistance proteins

Planten worden continu blootgesteld aan ziekteverwekkers. Om zich hiertegen te verdedigen bevatten planten onder meer eiwitten die ‘op wacht staan’ en onregelmatigheden in de cel kunnen detecteren die door ziekteverwekkers worden veroorzaakt. Deze eiwitten worden resistentie-eiwitten (R-eiwitten) genoemd. Zodra een R-eiwit een ziekteverwekker waarneemt wordt een sterke afweerreactie opgewekt. Ewa Lukasik-Shreepaathy deed onderzoek naar R-eiwitten die zogeheten NB-LRR-domeinen bevatten (NB-LRR staat voor nucleotide binding and leucine rich repeat). Deze NB-LRR-eiwitten fungeren als moleculaire schakelaars die plantafweer aan en uit kunnen zetten. Lukasik-Shreepaathy beschrijft de intra- en intermoleculaire interacties van een aantal R-eiwitten en keek naar de rol van specifieke R-eiwitdomeinen in het functioneren van het eiwit. Ze laat onder andere zien dat een R-eiwit uit gerst, MLA27, een stabiele binding vormt met ADP (chemische verbinding die betrokken is bij energiehuishouding). Dit ondersteunt het idee dat R-eiwitten functioneren als aan-uitschakelaars

Dual regulatory roles of the extended N-terminus for activation of the tomato Mi-1.2 resistance protein

Plant resistance (R) proteins mediate race-specific immunity and initiate host defenses that are often accompanied by a localized cell-death response. Most R proteins belong to the NB-LRR protein family as they carry a central NB-ARC domain fused to an LRR domain. The CC domain at the N-terminus of some Solanaceous NB-LRR proteins is extended with a solanaceae domain (SD). Tomato Mi-1.2, which confers resistance against nematodes, white flies, psyllids and aphids, encodes a typical SD-CNL protein. Here, we analyzed the role of the extended N-terminus for Mi-1.2 activation. Removal of the first part of the N-terminus (Nt1) induced Mi-1.2-mediated cell death that could be suppressed by over-expression of the second half of the N-terminal region (Nt2). Yet, autoactivating NB-ARC-LRR mutants require in trans co-expression of the N-terminal region to induce cell death, indicating that the N-terminus functions both as a negative and a positive regulator. Based on secondary structure predictions we could link both functions to three distinct subdomains; a typical CC domain and two novel, structurally-conserved helical subdomains called SD1 and SD2. A negative regulatory function could be assigned to the SD1 whereas SD2 and the CC together function as positive regulators of Mi-1.2 mediated cell deat