No abstract

Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here, we report that a conserved SNW/Ski-interacting protein (SKIP) domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in posttranscriptional regulation of circadian clock genes in Arabidopsis thaliana. Mutation in SKIP lengthens the circadian period in a temperature-sensitive manner and affects light input and the sensitivity of the clock to light resetting. SKIP physically interacts with the spliceosomal splicing factor Ser/Arg-rich protein45 and associates with the pre-mRNA of clock genes, such as PSEUDORESPONSE REGULATOR7 (PRR7) and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 59 and 39 splice donor and acceptor sites. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock function at a posttranscriptional level.

The tomato Mi-1 gene confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum eluphorbiae), and whiteflies (Bemisia tabaci and B. tabaci biotype B). Resistance to potato aphid is developmentally regulated and is not associated with induction of a hypersensitive response. The NahG transgene that eliminates endogenous salicylic acid (SA) was used to test the role of the SA signaling pathway in the resistance mediated by Mi-1 to potato aphids. Aphids survived longer on NahG tomato plants than on wild type. However, aphid reproduction was not affected on NahG tomato. Aphid resistance in Mi-1 NahG plants was completely abolished and the phenotype was successfully rescued by application of BTH (benzo(1,2,3)-thiaiazole-7-carbothioic acid S-methyl ester), indicating that the SA signaling pathway is an important component of Mi-1-mediated aphid resistance. Using virus-induced gene silencing, one or more mitogen-activated protein kinase (MAPK) cascades required for Mi-1-mediated aphid resistance were identified. Silencing plants for MAPK kinase (LeMKK2) and MAPKs (LeMPK2 and LeMPK1, or LeMPK3) resulted in attenuation of Mi-1-mediated aphid resistance. These results further demonstrate that resistance gene-mediated signaling events against piercing-sucking insects are similar to those against other plant pathogens.

The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.

Responses of resistant (Mi-1/Mi-1) and susceptible (mi-1/ mi-1) tomato (Solanum lycopersicum) to root-knot nematodes (RKNs; Meloidogyne spp.) infection were monitored using cDNA microarrays, and the roles of salicylic acid (SA) and jasmonic acid (JA) defense signaling were evaluated in these interactions. Array analysis was used to compare transcript profiles in incompatible and compatible interactions of tomato roots 24 h after RKN infestation. The jai1 and def1 tomato mutant, altered in JA signaling, and tomato transgenic line NahG, altered in SA signaling, in the presence or absence of the RKN resistance gene Mi-1, were evaluated. The array analysis identified 1,497 and 750 genes differentially regulated in the incompatible and compatible interactions, respectively. Of the differentially regulated genes, 37% were specific to the incompatible interactions. NahG affected neither Mi-1 resistance nor basal defenses to RKNs. However, jai1 reduced tomato susceptibility to RKNs while not affecting Mi-1 resistance. In contrast, the def1 mutant did not affect RKN susceptibility. These results indicate that JA-dependent signaling does not play a role in Mi-1-mediated defense; however, an intact JA signaling pathway is required for tomato susceptibility to RKNs. In addition, low levels of SA might be sufficient for basal and Mi-1 resistance to RKNs.