Calcium signaling: an emerging player in plant antiviral defense.

Calcium is a universal messenger in different kingdoms of living organisms and regulates most physiological processes, including defense against pathogens. The threat of viral infections in humans has become very clear in recent years, and this has triggered detailed research into all aspects of host-virus interactions, including the suppression of calcium signaling in infected cells. At the same time, however, the threat of plant viral infections is underestimated in society, and research in the field of calcium signaling during plant viral infections is scarce. Here we highlight an emerging role of calcium signaling for antiviral protection in plants, in parallel with the known evidence from studies of animal cells. Obtaining more knowledge in this domain might open up new perspectives for future crop protection and the improvement of food security.

Viral protein suppresses oxidative burst and salicylic acid-dependent autophagy and facilitates bacterial growth on virus-infected plants.

Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection.

Interactions of Rice tungro bacilliform pararetrovirus and its protein P4 with plant RNA-silencing machinery.

Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silencing, which correlated with reduced 21-nt siRNA levels and increased 22-nt siRNA levels. Our findings imply that RTBV generates decoy dsRNA that restricts siRNA production to the structured noncoding region and thereby protects other regions of the viral genome from repressive action of siRNAs, while the viral protein P4 interferes with cell-to-cell spread of antiviral silencing.

Silencing and innate immunity in plant defense against viral and non-viral pathogens.

The frontline of plant defense against non-viral pathogens such as bacteria, fungi and oomycetes is provided by transmembrane pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). To counteract this innate defense, pathogens deploy effector proteins with a primary function to suppress PTI. In specific cases, plants have evolved intracellular resistance (R) proteins detecting isolate-specific pathogen effectors, leading to effector-triggered immunity (ETI), an amplified version of PTI, often associated with hypersensitive response (HR) and programmed cell death (PCD). In the case of plant viruses, no conserved PAMP was identified so far and the primary plant defense is thought to be based mainly on RNA silencing, an evolutionary conserved, sequence-specific mechanism that regulates gene expression and chromatin states and represses invasive nucleic acids such as transposons. Endogenous silencing pathways generate 21-24 nt small (s)RNAs, miRNAs and short interfering (si)RNAs, that repress genes post-transcriptionally and/or transcriptionally. Four distinct Dicer-like (DCL) proteins, which normally produce endogenous miRNAs and siRNAs, all contribute to the biogenesis of viral siRNAs in infected plants. Growing evidence indicates that RNA silencing also contributes to plant defense against non-viral pathogens. Conversely, PTI-based innate responses may contribute to antiviral defense. Intracellular R proteins of the same NB-LRR family are able to recognize both non-viral effectors and avirulence (Avr) proteins of RNA viruses, and, as a result, trigger HR and PCD in virus-resistant hosts. In some cases, viral Avr proteins also function as silencing suppressors. We hypothesize that RNA silencing and innate immunity (PTI and ETI) function in concert to fight plant viruses. Viruses counteract this dual defense by effectors that suppress both PTI-/ETI-based innate responses and RNA silencing to establish successful infection.

Primary and secondary siRNAs in geminivirus-induced gene silencing.

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.

Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis.

Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA. This tasiRNA and miR173 guide Argonaute 1 complexes to excise the segments from TAS2 and three TAS1 transcripts including TAS1c itself to be converted to dsRNAs, which restricts siRNA production to a region between the two cleavage sites. TAS1c is also feedback regulated by a cis-acting siRNA. We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation. TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs.

Pol II-directed short RNAs suppress the nuclear export of mRNA.

The synthesis and subsequent nuclear export of non-coding RNA (ncRNA) directed by RNA polymerase (Pol) II is very sensitive to abiotic and biotic external stimuli including pathogen challenges. To assess whether stress-induced ncRNAs may suppress the nuclear export of mRNA, we exploited the ability of Agrobacterium tumefaciens to co-deliver Pol I, II and III promoter-based vectors for the transcription of short (s) ncRNAs, GFP mRNA or genomic RNA of plant viruses (Tobacco mosaic virus, TMV; or Potato virus X, PVX) into the nucleus of Nicotiana benthamiana cells. We showed that, in contrast to Pol I- and Pol III-derived sncRNAs, all tested Pol II-derived sncRNAs (U6 RNA, tRNA or artificial RNAs) resulted in decreased expression of GFP and host mRNA. The level of this inhibitory effect depended on the non-coding transcript length and promoter strength. Short coding RNA (scRNA) can also compete with mRNA for nuclear export. We showed that scRNA, an artificial 117-nt short sequence encoding Elastin-Like peptide element tandems with FLAG sequence (ELF) and the 318-nt N. benthamiana antimicrobial peptide thionin (defensin) gene efficiently decreased GFP expression. The stress-induced export of Pol II-derived sncRNA and scRNA into the cytoplasm via the mRNA export pathway may block nucleocytoplasmic traffic including the export of mRNA responsible for antivirus protection. Consistent with this model, we observed that Pol II-derived sncRNAs as well as scRNA, thionin and ELF strongly enhanced the cytoplasmic reproduction of TMV and PVX RNA.

Superexpression of tuberculosis antigens in plant leaves.

Recent developments in genetic engineering allow the employment of plants as factories for 1/foreign protein production. Thus, tuberculosis (TB) ESAT6 antigen was expressed in different plant systems, but the level of vaccine protein accumulation was extremely low. We describe the technology for superexpression of TB vaccine proteins (Ag85B, ESAT6, and ESAT6:Ag85B fusion) in plant leaves which involves: (i) construction of tobacco mosaic virus-based vectors with the coat protein genes substituted by those for TB antigens; (ii) Agrobacterium-mediated delivery to plant leaf tissues of binary vectors containing the cDNA copy of the vector virus genome; and (iii) replication of virus vectors in plant cells under conditions suppressing the virus-induced gene silencing. This technology enables efficient production of the TB vaccine proteins in plants; in particular, the level of Ag85B antigen accumulation was not less than 800 mg/kg of fresh leaves. Expression of TB antigens in plant cells as His(6)-tagged proteins promoted their isolation and purification by Ni-NTA affinity chromatography. Deletion of transmembrane domains from Ag85B caused a dramatic increase in its intracellular stability. We propose that the strategy of TB antigens superproduction in a plant might be used as a basis for the creation of prophylactic and therapeutic vaccine against TB.