Environmental RNAi in herbivorous insects.

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism.

Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation.

Both plants and animals require the activity of proteins containing nucleotide binding (NB) domain and leucine-rich repeat (LRR) domains for proper immune system function. NB-LRR proteins in plants (NLR proteins in animals) also require conserved regulation via the proteins SGT1 and cytosolic HSP90. RAR1, a protein specifically required for plant innate immunity, interacts with SGT1 and HSP90 to maintain proper NB-LRR protein steady-state levels. Here, we present the identification and characterization of specific mutations in Arabidopsis HSP90.2 that suppress all known phenotypes of rar1. These mutations are unique with respect to the many mutant alleles of HSP90 identified in all systems in that they can bypass the requirement for a cochaperone and result in the recovery of client protein accumulation and function. Additionally, these mutations separate HSP90 ATP hydrolysis from HSP90 function in client protein folding and/or accumulation. By recapitulating the activity of RAR1, these novel hsp90 alleles allow us to propose that RAR1 regulates the physical open-close cycling of a known "lid structure" that is used as a dynamic regulatory HSP90 mechanism. Thus, in rar1, lid cycling is locked into a conformation favoring NB-LRR client degradation, likely via SGT1 and the proteasome.

Type III effector activation via nucleotide binding, phosphorylation, and host target interaction.

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.

Temperature-induced reversible conformational change in the first 100 residues of alpha-synuclein.

Natively disordered proteins are a growing class of anomalies to the structure-function paradigm. The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10 degrees C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing alpha-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra obtained at 35 degrees C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of alpha-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions.

Macromolecular crowding in the Escherichia coli periplasm maintains alpha-synuclein disorder.

The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. Most studies of this protein are performed in dilute solution, but its biologically relevant role is performed in the crowded environment inside cells. We addressed the effects of macromolecular crowding on alpha-synuclein by combining NMR data acquired in living Escherichia coli with in vitro NMR data. The crowded environment in the E.coli periplasm prevents a conformational change that is detected at 35 degrees C in dilute solution. This change is associated with an increase in hydrodynamic radius and the formation of secondary structure in the N-terminal 100 amino acid residues. By preventing this temperature-induced conformational change, crowding in the E.coli periplasm stabilizes the disordered monomer. We obtain the same stabilization in vitro upon crowding alpha-synuclein with 300 g/l of bovine serum albumin, indicating that crowding alone is sufficient to stabilize the disordered, monomeric protein. Two disease-associated variants (A30P and A53T) behave in the same way in both dilute solution and in the E.coli periplasm. These data reveal the importance of approaching the effects of macromolecular crowding on a case-by-case basis. Additionally, our work shows that discrete structured protein conformations may not be achieved by alpha-synuclein inside cells, implicating the commonly overlooked aspect of macromolecular crowding as a possible factor in the etiology of Parkinson's disease.

Viral genetic determinants for thrips transmission of Tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) is transmitted exclusively by thrips in nature. A reassortment-based viral genetic system was used to map transmissibility by thrips to the medium (M) RNA of TSWV. To locate determinants of thrips transmission in the M RNA, 30 single-lesion isolates (SLIs) were generated from a single TSWV isolate that was inefficiently transmitted by thrips. Three of the 30 SLIs were transmitted by thrips, and 27 were not. Sequence analysis of the M RNA, thrips transmissibility assays, G(C) protein analysis, and transmission electron microscopic studies revealed that a specific nonsynonymous mutation (C1375A) in the G(N)/G(C) ORF of the M RNA resulted in the loss of thrips transmissibility without inhibition of virion assembly. This was in contrast to other nontransmissible SLIs, which had frameshift and/or nonsense mutations in the G(N)/G(C) ORF but were defective in virion assembly. The G(C) glycoprotein was detectable in the C1375A mutants but not in the frameshift or nonsense mutants. We report a specific viral determinant associated with virus transmission by thrips. In addition, the loss of transmissibility was associated with the accumulation of defective haplotypes in the population, which are not transmissible by thrips, rather than with the presence of a dominant haplotype that is inefficiently transmitted by thrips. These results also indicate that the glycoproteins may not be required for TSWV infection of plant hosts but are required for transmissibility by thrips.