(1) and found widespread application in plant virology either as tags for specific viral proteins during infection or as general reporters of cell infection (2, 3). While the study of plant viruses has benefited greatly from the use of GFP, it is known that FP-expressing viruses exhibit reduced infection efficiency that can negatively affect host range relative to the wild-type virus (4-6). The same is true for animal viruses, especially when FPs are attached to structural components of tightly packed virions (7). Moreover, the increased genetic load of plant viruses carrying a FP severely limits both local and systemic spread compared with that of the wild-type virus (6). Attempts have been made to overcome this problem in the extensively studied positive-strand RNA virus, Tobacco mosaic virus (TMV), through molecular evolution of the viral movement protein (MP), a protein critical for cell-tocell movement of the viral genome (6). An alternative strategy would be to utilize a smaller FP to reduce the genetic load. However, generation of smaller derivatives from GFP-based FPs is unlikely because the -barrel structure of the protein is intrinsic to its function (8).Fluorescent peptide ligands represent promising smaller alternatives to GFP-based FPs (9). Yet, the necessity for an exogenous chemical substrate introduces limitations for this approach, particularly in plants where the cell wall poses an additional barrier to permeability (10), prompting us to search for other genetically encoded candidates. The utility of linear tetrapyrrole (bilin)-binding proteins as fluorescent probes in the near infrared region of the spectrum has been recognized (11), as has the potential for flavin-based fluorescent proteins as in vivo reporters (12). The latter are derived from photosensory modules known as light, oxygen or voltage sensing (LOV) domains present in a diverse range of photoreceptors from bacteria, fungi, and plants (13,14). UV/blue light is detected via the chromophore flavin mononucleotide (FMN) located within the LOV domain, giving the protein a weak intrinsic fluorescence with a maximal emission wavelength at 495 nm (15). Although used successfully to monitor bacterial cell populations (12, 16), the suitability of LOV-based FPs for studying protein localization and trafficking has not been investigated.In the present study, we examined whether LOV-based FPs could be used as fluorescent reporters of virus infection in plant cells because their relatively small size offers an advantage over GFP. Through the molecular evolution of plant-derived LOV domains, we have isolated a photoreversible FP that can be used effectively to track protein distribution within living cells. This FP, termed iLOV, outperformed GFP as a reporter of plant virus infection and movement, and conferred improved functionality over GFP when fused to proteins required for virus spread. iLOV, therefore, represents a new genetically encoded alternative to GFP-based FPs that exhibits greater utility for monitoring virus infection. Results a...
Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.
Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.
In plants, as in animals, programmed cell death (PCD) is a key process responsible for the elimination of unneeded structures and for overall shape remodeling during development [1]; however, the molecular mechanisms remain poorly understood. Despite the absence of canonical caspases in plants, dying plant cells show an increased proteolytic caspase-like activity [2]. Moreover, the cell death can be suppressed using synthetic [2] or natural [3] caspase inhibitors. This raises the question of whether plants have specific cysteine proteases with a role similar to metazoan caspases in the execution of PCD. Metacaspases are the best candidates to perform this role, because they contain a caspasespecific catalytic diad of histidine and cysteine as well as conserved caspase-like secondary structure [4,5]. Here we show the first experimental evidence for metacaspase function in the activation and/or execution of PCD in plants, and also demonstrate the fundamental requirement of plant metacaspase for embryogenesis.We explored the role of plant metacaspases in PCD using a model system of somatic embryogenesis of Norway spruce (Picea abies), where the pathway of embryo development (Figure 1A) resembles zygotic embryogeny, even though the embryo origin is different in each case (i.e., somatic cells in proembryogenic mass
Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19–40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.
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