The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.

Summary: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is one of the most devastating diseases in rice worldwide. The dominant resistance gene, Pi‐d2 [previously named Pi‐d(t)2], present in the rice variety Digu, confers gene‐for‐gene resistance to the Chinese blast strain, ZB15. Pi‐d2 was previously mapped close to the centromere of chromosome 6. In this study, the Pi‐d2 gene was isolated by a map‐based cloning strategy. Pi‐d2 encodes a receptor‐like kinase protein with a predicted extracellular domain of a bulb‐type mannose specific binding lectin (B‐lectin) and an intracellular serine–threonine kinase domain. Pi‐d2 is a single‐copy gene that is constitutively expressed in the rice variety Digu. Transgenic plants carrying the Pi‐d2 transgene confer race‐specific resistance to the M. grisea strain, ZB15. The Pi‐d2 protein is plasma membrane localized. A single amino acid difference at position 441 of Pi‐d2 distinguishes resistant and susceptible alleles of rice blast resistance gene Pi‐d2. Because of its novel extracellular domain, Pi‐d2 represents a new class of plant resistance genes.

Arabidopsis NONHOST1 ( NHO1 ) is required for limiting the in planta growth of nonhost Pseudomonas bacteria but completely ineffective against the virulent bacterium Pseudomonas syringae pv. tomato DC3000. However, the molecular basis underlying this observation remains unknown. Here we show that NHO1 is transcriptionally activated by flagellin. The nonhost bacterium P. syringae pv. tabaci lacking flagellin is unable to induce NHO1 , multiplies much better than does the wild-type bacterium, and causes disease symptoms on Arabidopsis . DC3000 also possesses flagellin that is potent in NHO1 induction, but this induction is rapidly suppressed by DC3000 in a type III secretion system-dependent manner. Direct expression of DC3000 effectors in protoplasts indicated that at least nine effectors, HopS1, HopAI1, HopAF1, HopT1-1, HopT1-2, HopAA1-1, HopF2, HopC1, and AvrPto, are capable of suppressing the flagellin-induced NHO1 expression. One of the effectors, HopAI1, is conserved in both animal and plant bacteria. When expressed in transgenic Arabidopsis plants, HopAI1 promotes growth of the nonpathogenic hrpL ^- mutant bacteria. In addition, the purified phytotoxin coronatine, a known virulence factor of P. syringae , suppresses the flagellin-induced NHO1 transcription. These results demonstrate that flagellin-induced defenses play an important role in nonhost resistance. A remarkable number of DC3000 virulence factors act in the plant cell by suppressing the species level defenses, and that contributes to the specialization of DC3000 on Arabidopsis .