Perturbation of nuclear-cytosolic shuttling of Rx1 compromises extreme resistance and translational arrest of potato virus X transcripts.

Many plant intracellular immune receptors mount a hypersensitive response (HR) upon pathogen perception. The concomitant localized cell death is proposed to trap pathogens, such as viruses, inside infected cells, thereby preventing their spread. Notably, extreme resistance (ER) conferred by the potato immune receptor Rx1 to potato virus X (PVX) does not involve the death of infected cells. It is unknown what defines ER and how it differs from HR-based resistance. Interestingly, Rx1 can trigger an HR, but only upon artificial (over)expression of PVX or its avirulence coat protein (CP). Rx1 has a nucleocytoplasmic distribution and both pools are required for HR upon transient expression of a PVX-GFP amplicon. It is unknown whether mislocalized Rx1 variants can induce ER upon natural PVX infection. Here, we generated transgenic Nicotiana benthamiana producing nuclear- or cytosol-restricted Rx1 variants. We found that these variants can still mount an HR. However, nuclear- or cytosol-restricted Rx1 variants can no longer trigger ER or restricts viral infection. Interestingly, unlike the mislocalized Rx1 variants, wild-type Rx1 was found to compromise CP protein accumulation. We show that the lack of CP accumulation does not result from its degradation but is likely to be linked with translational arrest of its mRNA. Together, our findings suggest that translational arrest of viral genes is a major component of ER and, unlike the HR, is required for resistance to PVX.

Unlike Many Disease Resistances, Rx1-Mediated Immunity to Potato Virus X Is Not Compromised at Elevated Temperatures.

Specificity in the plant immune system is mediated by Resistance (R) proteins. Most R genes encode intracellular NLR-type immune receptors and these pathogen sensors require helper NLRs to activate immune signaling upon pathogen perception. Resistance conferred by many R genes is temperature sensitive and compromised above 28°C. Many Solanaceae R genes, including the potato NLR Rx1 conferring resistance to Potato Virus X (PVX), have been reported to be temperature labile. Rx1 activity, like many Solanaceae NLRs, depends on helper-NLRs called NRC's. In this study, we investigated Rx1 resistance at elevated temperatures in potato and in Nicotiana benthamiana plants stably expressing Rx1 upon rub-inoculation with GFP-expressing PVX particles. In parallel, we used susceptible plants as a control to assess infectiousness of PVX at a range of different temperatures. Surprisingly, we found that Rx1 confers virus resistance in N. benthamiana up to 32°C, a temperature at which the PVX::GFP lost infectiousness. Furthermore, at 34°C, an Rx1-mediated hypersensitive response could still be triggered in N. benthamiana upon PVX Coat-Protein overexpression. As the Rx1-immune signaling pathway is not temperature compromised, this implies that at least one N. benthamiana helper NRC and its downstream signaling components are temperature tolerant. This finding suggests that the temperature sensitivity for Solanaceous resistances is likely attributable to the sensor NLR and not to its downstream signaling components.

Activation of immune receptor Rx1 triggers distinct immune responses culminating in cell death after 4 hours.

Intracellular nucleotide-binding leucine-rich repeat (NLR)-type immune receptors are a fundamental part of plant immune systems. As infection occurs at foci, activation of immune responses is typically non-uniform and non-synchronized, hampering the systematic dissection of their cellular effects and determining their phasing. We investigated the potato NLR Rx1 using the CESSNA (Controlled Expression of effectors for Synchronized and Systemic NLR Activation) platform. CESSNA-mediated Potato virus X coat protein (CP) expression allowed the monitoring of Rx1-mediated immune responses in a quantitative and reproducible manner. Rx1 was found to trigger a reactive oxygen species (ROS) burst and ion leakage within 1 h and a change in autofluorescence within 2 h after the induction of CP production. After 2 h, HIN1 expression was increased and single-stranded DNA (ssDNA) damage and loss of cellular integrity became apparent, followed by double-stranded DNA (dsDNA) damage after 3 h and increased PR-1a, LOX, ERF1 and AOX1B expression and cell death at 4 h. Nuclear exclusion of Rx1 resulted in increased basal levels of ROS and permitted Rx1 activation by an Rx1-breaking CP variant. In contrast, nuclear-targeted Rx1 showed diminished basal ROS levels, and only avirulent CP could trigger a compromised ROS production. Both nuclear-excluded and nuclear-targeted Rx1 triggered a delayed ion leakage compared with non-modified Rx1, suggesting that ion leakage and ROS production originate from distinct signalling pathways. This work offers novel insights into the influence of Rx1 localization on its activity, and the interplay between Rx1-triggered processes.

Trans-kingdom cross-talk: small RNAs on the move.

This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. However, they may only represent the tip of the iceberg, since the emerging picture suggests that organisms in biological niches commonly exchange RNA-silencing signals. In this case, we need to take this into account fully to understand how a given biological equilibrium is obtained. Despite many observations of trans-kingdom RNA signal transfer, several mechanistic aspects of these signals remain unknown. Such RNA signal transfer is already being exploited for practical purposes, though. Pathogen genes can be silenced by plant-produced sRNAs designed to affect these genes. This is also known as Host-Induced Genes Silencing (HIGS), and it has the potential to become an important disease-control method in the future.

DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues.

BACKGROUND: DAYSLEEPER is a domesticated transposase that is essential for development in Arabidopsis thaliana [Nature, 436:282-284, 2005]. It is derived from a hAT-superfamily transposon and contains many of the features found in the coding sequence of these elements [Nature, 436:282-284, 2005, Genetics, 158:949-957, 2001]. This work sheds light on the expression of this gene and localization of its product in protoplasts and in planta. Using deletion constructs, important domains in the protein were identified. RESULTS: DAYSLEEPER is predominantly expressed in meristems, developing flowers and siliques. The protein is mainly localized in the nucleus, but can also be seen in discrete foci in the cytoplasm. Using several vesicular markers, we found that these foci belong to vesicular structures of the trans-golgi network, multivesicular bodies (MVB's) and late endosomes. The central region as well as both the N- and the C-terminus are essential to DAYSLEEPER function, since versions of DAYSLEEPER deleted for these regions are not able to complement the daysleeper phenotype. Like hAT-transposases, we show that DAYSLEEPER has a functionally conserved dimerization domain [J Biol Chem, 282:7563-7575, 2007]. CONCLUSIONS: DAYSLEEPER has retained the global structure of hAT transposases and it seems that most of these conserved features are essential to DAYSLEEPER's cellular function. Although structurally similar, DAYSLEEPER seems to have broadened its range of action beyond the nucleus in comparison to transposases.

The SLEEPER genes: a transposase-derived angiosperm-specific gene family.

BACKGROUND: DAYSLEEPER encodes a domesticated transposase from the hAT-superfamily, which is essential for development in Arabidopsis thaliana. Little is known about the presence of DAYSLEEPER orthologs in other species, or how and when it was domesticated. We studied the presence of DAYSLEEPER orthologs in plants and propose a model for the domestication of the ancestral DAYSLEEPER gene in angiosperms. RESULTS: Using specific BLAST searches in genomic and EST libraries, we found that DAYSLEEPER-like genes (hereafter called SLEEPER genes) are unique to angiosperms. Basal angiosperms as well as grasses (Poaceae) and dicotyledonous plants possess such putative orthologous genes, but SLEEPER-family genes were not found in gymnosperms, mosses and algae. Most species contain more than one SLEEPER gene. All SLEEPERs contain a C2H2 type BED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the BED-zinc finger and dimerization domain, which are hallmark features in the SLEEPER family. Although SLEEPER genes are structurally conserved between species, constructs with SLEEPER genes from grapevine and rice did not complement the daysleeper phenotype in Arabidopsis, when expressed under control of the DAYSLEEPER promoter. However these constructs did cause a dominant phenotype when expressed in Arabidopsis. Rice plant lines with an insertion in the RICESLEEPER1 or 2 locus displayed phenotypic abnormalities, indicating that these genes are functional and important for normal development in rice. We suggest a model in which we hypothesize that an ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution. Evidence is also presented for more recent retroposition events of SLEEPER genes, such as an event in the rice genome, which gave rise to the RICESLEEPER1 and 2 genes. CONCLUSIONS: We propose the ancestral SLEEPER gene was formed after a process of retro-transposition during the evolution of the first angiosperms. It may have acquired an important function early on, as mutation of two SLEEPER genes in rice, like the daysleeper mutant in A. thaliana gave a developmental phenotype indicative of their importance for normal plant development.

DNA methylation and normal chromosome behavior in Neurospora depend on five components of a histone methyltransferase complex, DCDC.

Methylation of DNA and of Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, and fungi. In Neurospora crassa, methylation of H3K9 by DIM-5 directs cytosine methylation by recruiting a complex containing Heterochromatin Protein-1 (HP1) and the DIM-2 DNA methyltransferase. We report genetic, proteomic, and biochemical investigations into how DIM-5 is controlled. These studies revealed DCDC, a previously unknown protein complex including DIM-5, DIM-7, DIM-9, CUL4, and DDB1. Components of DCDC are required for H3K9me3, proper chromosome segregation, and DNA methylation. DCDC-defective strains, but not HP1-defective strains, are hypersensitive to MMS, revealing an HP1-independent function of H3K9 methylation. In addition to DDB1, DIM-7, and the WD40 domain protein DIM-9, other presumptive DCAFs (DDB1/CUL4 associated factors) co-purified with CUL4, suggesting that CUL4/DDB1 forms multiple complexes with distinct functions. This conclusion was supported by results of drug sensitivity tests. CUL4, DDB1, and DIM-9 are not required for localization of DIM-5 to incipient heterochromatin domains, indicating that recruitment of DIM-5 to chromatin is not sufficient to direct H3K9me3. DIM-7 is required for DIM-5 localization and mediates interaction of DIM-5 with DDB1/CUL4 through DIM-9. These data support a two-step mechanism for H3K9 methylation in Neurospora.

Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.

Chromocenters in Arabidopsis thaliana are discrete nuclear domains of mainly pericentric heterochromatin. They are characterized by the presence of repetitive sequences, methylated DNA and dimethylated histone H3K9. Here we show that dedifferentiation of specialized mesophyll cells into undifferentiated protoplasts is accompanied by the disruption of chromocenter structures. The dramatic reduction of heterochromatin involves the decondensation of all major repeat regions, also including the centromeric 180 bp tandem repeats. Only the 45S rDNA repeat remained in a partly compact state in most cells. Remarkably, the epigenetic indicators for heterochromatin, DNA methylation and H3K9 dimethylation, did not change upon decondensation. Furthermore, the decondensation of pericentric heterochromatin did not result in transcriptional reactivation of silent genomic elements. The decondensation process was reversible upon prolonged culturing. Strikingly, recondensation of heterochromatin into chromocenters is a stepwise process. Compaction of the tandemly arranged 45S rDNA regions occurs first, followed by the centromeric 180 bp and the 5S rDNA repeats and finally the dispersed repeats, including transposons. The sequence of reassembly seems to be correlated to the size of the repeat domains. Our results indicate that different types of pericentromeric repeats form different types of heterochromatin, which subsequently merge to form a chromocenter.