Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation.     

Synthesis and uptake of cytoplasmically synthesized pyruvate, pi dikinase polypeptide by chloroplasts

Polyadenylated RNA was isolated from maize leaves and translated in vitro. In agreement with a previous report by others, we found among the translation products a 110-kilodalton pyruvate orthophosphate dikinase (PPDK) precursor that is about 16 kilodaltons larger than the polypeptide isolated from cells. This maize PPDK precursor polypeptide was taken up from the translation product mixture by intact spinach chloroplasts and yielded a mature PPDK polypeptide (94 kilodaltons). The uptake and processing support the proposal that the extra 16-kilodalton size of the polypeptide from in vitro translation of maize leaf mRNA represents a transit sequence which is cleaved after its entry into chloroplasts. Moreover, these results provide additional evidence that in vivo in maize leaf cells PPDK polypeptide is synthesized in the cytoplasm and is transported into the chloroplasts.

Location of PPDK in C₃ plant leaves was investigated by immunochemical analysis. Intact chloroplasts were isolated from leaves of spinach, wheat, and maize. A protein blot of stromal protein in each case gave rise to bands corresponding to authentic PPDK polypeptide. This result indicates that PPDK is present in chloroplasts of C₃ plant leaves as it is in the case of C₄ plants.

     

Silencing of aphid genes by dsRNA feeding from plants

Background

RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.

Methodology/Principal Findings

In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.

Conclusions/Significance

Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.     

The efficiency of RNA interference for conferring stable resistance to plum pox virus

Plum transformed with an intron hairpin RNA CP (ihpRNA-CP) was resistant to plum pox virus (PPV) infection through the specific process of RNA silencing involving both small interfering-RNA (siRNA) and a methylated virus transgene. Silencing specifically targeted the PPV genome and led to the degradation of viral RNA in the model plant species Nicotiana benthamiana and the natural Prunus domestica host. Plums inoculated with the five major PPV strains, three widespread PPV strains (D, M, and Rec), and the atypical EA strain did not allow systemic spread of PPV in greenhouse-grown transgenic ihRNA-CP plum over multiple cycles of vegetative growth and cold-induced dormancy. PPV ihRNA-CP N. benthamiana displayed an immunity reaction and also allowed for the testing of PPV-C, a strain that was unable to infect P. domestica. This stable resistance demonstrated in plum based on the accumulation of siRNA can prevent PPV infection and can also act as a “curative” when PPV is inoculated through graft inoculation, through a recovery reaction. Regardless PPV strain variability based on geography, host species, epidemiology and serotypes of the CP protein and substitutions of nucleotides at the NH₂-terminus of CP of the major five PPV strains tested, we show that the use of a PPV-CP intron hairpin (ihp) RNA is an effective strategy to specifically target the PPV genome. We provide methods and tools that demonstrate a reliable path towards developing PPV resistance suitable for protecting stone fruit orchards.     

Sheet-like clay nanoparticles deliver RNA into developing pollen to efficiently silence a target gene

Topical application of double-stranded RNA (dsRNA) can induce RNA interference (RNAi) and modify traits in plants without genetic modification. However, delivering dsRNA into plant cells remains challenging. Using developing tomato (Solanum lycopersicum) pollen as a model plant cell system, we demonstrate that layered double hydroxide (LDH) nanoparticles up to 50 nm in diameter are readily internalized, particularly by early bicellular pollen, in both energy-dependent and energy-independent manners and without physical or chemical aids. More importantly, these LDH nanoparticles efficiently deliver dsRNA into tomato pollen within 2–4 h of incubation, resulting in an 89% decrease in transgene reporter mRNA levels in early bicellular pollen 3-d post-treatment, compared with a 37% decrease induced by the same dose of naked dsRNA. The target gene silencing is dependent on the LDH particle size, the dsRNA dose, the LDH–dsRNA complexing ratio, and the treatment time. Our findings indicate that LDH nanoparticles are an effective nonviral vector for the effective delivery of dsRNA and other biomolecules into plant cells.

Developing tomato pollen internalizes layered double hydroxide nanoparticles smaller than 50 nm that facilitate delivery of double-stranded RNA, enhancing RNA interference of a target gene.     

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.     

Atypical Protein Kinase Cι Is Required for Wnt3a-dependent Neurite Outgrowth and Binds to Phosphorylated Dishevelled 2

Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor cell lines was mediated by Frizzled3, Dishevelled (Dvl), and c-Jun N-terminal kinase (Endo, Y., Beauchamp, E., Woods, D., Taylor, W. G., Toretsky, J. A., Uren, A., and Rubin, J. S. (2008) Mol. Cell. Biol. 28, 2368–2379). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1δ, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer, Y. E., and Rubin, J. S. (2011) J. Cell Biol. 192, 993–1004). However, the functional relevance of Dvl phosphorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCι but not PKCζ. Wnt3a stimulated PKCι activation and caused a punctate distribution of pPKCι in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCι expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCι/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKCι and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCι, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (González-Sancho, J. M., Greer, Y. E., Abrahams, C. L., Takigawa, Y., Baljinnyam, B., Lee, K. H., Lee, K. S., Rubin, J. S., and Brown, A. M. (2013) J. Biol. Chem. 288, 9428–9437). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKCι. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.     

A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

Tomato bifunctional nuclease 1 (TBN1) is a polyfunctional protein with anticancerogenic activity originally isolated as an overexpressed protein from viroid-infected tomato. Its molecular farming in plant cells could be a non-expensive source for its biotechnology preparation. So we analysed TBN1 expression in Agrobacterium-infiltrated leaf sectors of Nicotiana benthamiana and in transformed suspension culture of tobacco BY-2 cells. During its transient expression, TBN1 mRNA was strongly degraded within a hot spot localized in the 3′ region. This early degradation process was inhibited by PTGS suppressors p19 and p38 resulting in increased TBN1 mRNA and protein yield. In parallel to degradation of TBN1 mRNA, high mRNA levels of two RNA-dependent RNA polymerases were detected in infiltrated leaf sectors, as well as in the transformed tobacco suspension culture BY-2, where low expression of the nuclease was stably maintained. Higher TBN1 mRNA and nuclease activity levels were found during its molecular farming in RDR6-deficient N. benthamiana plants. By fluorescent microscopy of infiltrated and transformed plant cells, the nuclease-GFP fusion protein was shown to be organized in filament-like structures.     

Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.     

RNA Synthesis in Phaseolus Chloroplasts: II. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLASTS FROM DEVELOPING AND SENESCING LEAVES

The rate of RNA synthesis in chloroplasts from the primary leavesof Phaseolus vulgaris L. cv. Canadian Wonder was measured invitro as plant age increased. The rate per leaf began to fallbefore the leaf was 70% expanded. At full expansion, activityhad fallen by 70%. Chloroplast RNA synthesis per unit chlorophyllwas falling before the leaf was 25% expanded. When all parts of the plant above the mature primary leaveswere removed (detopping) chloroplast RNA synthesis in theseleaves rose within 36 h. The rate increased to a maximum 3–4d after detopping, when it was 5–10 times control values;thereafter it fell again. The chlorophyll content began to increaseabout 4 d after detopping, eventually rising by 100%. Detoppingcaused a 3-fold increase in the Triton X-100-soluble DNA contentof chloroplast preparations, measured after 3.5 d. At that timethe rate of RNA synthesis per unit Triton-soluble DNA was thesame in chloroplasts from the primary leaves of intact and detoppedplants. Detopping also resulted in an increase in the depthof the leaf palisade layer. The effects of detopping on chloroplasts were prevented by darknessand reduced by shading. Increased chloroplast RNA polymerase activity was also inducedin the primary leaves by placing a polythene bag over intactplants, enclosing everything above these leaves. Removal ofthe roots from detopped plants prevented the rise in the rateof chloroplast RNA synthesis.     

Adenovirus-mediated RNA interference against collagen-specific molecular chaperone 47-KDa heat shock protein suppresses scar formation on mouse wounds

It has been shown in many investigations that the abnormally increasing production and deposition of collagen is one of the important mechanisms of pathological scars and other fibrotic diseases [Wang Z, Inokuchi T, Nemoto TK, Uehara M, Baba TT. Antisense oligonucleotide against collagen-specific molecular chaperone 47-kDa heat shock protein suppresses scar formation in rat wounds. Plast Reconstr Surg 2003 May; 111(6):1980-7; Obayashi K, Akamatsu H, Okano Y, Matsunage K, Masaki H. Exogenous nitric oxide enhances the synthesis of type I collagen and heat shock protein 47 by normal human dermal fibroblasts. J Dermatol Sci 2006 Feb; 41(2): 121-6 [e pub. 2005 Sep 19]; Kakugawa T, Mukae H, Hayashi T, Ishii H, Nakayama S, Sakamoto N, et-al. Expression of HSP47 in usual interstitial pneumonia and nonspecific interstitial pneumonia. Respir Res 2005 Jun;14(6): 57; Razzaque MS, Taguchi T. The possible role of colligin/HSP47, a collagen-binding protein, in the pathogenesis of human and experimental fibrotic diseases. Histol Histopathol 1999 Oct; 14(4): 1199-1212; Sharp PA. RNA interference - 2001. Genes Dev 2001 Mar 1; 15(5): 485-90; Ohashi S, Abe H, Takahashi T, Yamamoto Y, Takeuchi M, Arai H, et-al. Advanced glycation end products increase collagen-specific chaperone protein in mouse diabetic nephropathy. J Biol Chem 2004 May 7; 279(19): 19816-23 [epub 2004 Mar 5]]. RNA interference is the process that double-stranded RNA induces the homology-dependent degradation of cognate mRNA mediated by 21-23 nucleotide short interfering RNA (siRNA). In this study, we investigated the effect of HSP47-specific siRNA on the fibroblast cells, and then constructed adenovirus containing siRNA against HSP47 to inhibit the formation of scar in animal model. In this pilot study, HSP47 was targeted by this vector. Our results showed that the HSP47-specific siRNA could inhibit the expression of HSP47 at the level of mRNA and protein. Furthermore, adenovirus-mediated transfer of siRNA against HSP47 could inhibit the expression of type I collagen and the formation of scar tissue in animal model. It is likely that the inhibition of HSP47 by RNA interference (RNAi) could be developed as a powerful approach to prevent the scar formation.     

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.     

Photosynthetic oxygen exchange in isolated cells and chloroplasts of c(3) plants

Photosynthetic O₂-production and photorespiratory O₂-uptake were measured, using stable isotope techniques, in isolated intact leaf cells of the C₃ plant Xanthium strumarium L., and isolated intact chloroplasts of Spinacia oleracea L (var. Yates 102). Considerable light dependent O₂-uptake was observed in both systems, a proportion of which could be suppressed by CO₂ (63% suppression in chloroplasts by 50 micromolar CO₂, 58% in cells by 100 micromolar CO₂ and 250 micromolar O₂). At low O₂, O₂-uptake was CO₂ insensitive. At high CO₂ up to 19% of total electron flow was to O₂ in cells and up to 14% in chloroplasts. O₂-uptake showed inhibition by KCN (61% in cells, 35% in chloroplasts by 0.2 millimolar KCN). O₂-uptake half saturated at 75 to 85 micromolar O₂ in cells and 50 to 65 micromolar O₂ in chloroplasts, at low CO₂. The results are discussed in terms of the RuP₂-oxygenase reaction and direct photoreduction of O₂ via a Mehler reaction.     

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.     

A nanoformulation of siRNA and its role in cancer therapy: In vitro and in vivo evaluation

Overexpression of anti-apoptotic Bcl-2 is often observed in a wide variety of human cancers. It prevents the induction of apoptosis in neoplastic cells and contributes to resistance to chemotherapy. RNA interference has emerged as an efficient and selective technique for gene silencing. The potential to use small interfering RNA (siRNA) as a therapeutic agent for the treatment of cancer has elicited a great deal of interest. However, insufficient cellular uptake and poor stability have limited its therapeutic applications. The purpose of this study was to prepare chitosan nanoparticles via ionic gelation of chitosan by tripolyphosphate for effective delivery of siRNA to silence the anti-apoptotic Bcl-2 gene in neoplastic cells. Chitosan nanoparticles loaded with siRNA were in the size range 190 to 340 nm with a polydispersive index ranging from 0.04 to 0.2. They were able to completely bind with siRNA, provide protection against nuclease degradation, and enhance the transfection. Cell culture studies revealed that nanoparticles with entrapped siRNA could efficiently silence the antiapoptotic Bcl-2 gene. Studies on Swiss albino mice showed that siRNA could be effectively delivered through nanoparticles. There was significant decrease in the tumor volume. Blocking the expression of anti-apoptotic Bcl-2 can enhance the sensitivity of cancerous cells to anti-cancer drugs and the apoptosis rate. Therefore, nanoformulations with siRNA can be promoted as an adjuvant therapy in combination with anti-cancer drugs.     

Dianthus caryophyllus stems and Zantedeschia aethiopica petioles/pedicels show anatomical features indicating efficient photosynthesis

The fine structure of the green stem of Dianthus caryophyllus, the leaf petiole and the flower pedicel of Zantedeschia aethiopica were studied using light and scanning electron microscopy. It was revealed that these non-foliar plant parts of both species possess epidermis with numerous stomata. Stomatal density of D. caryophyllus stem was found to be relatively high (79 vs 100 per mm² found on leaf surface). Z. aethiopica petioles and pedicels also possess numerous stomata (17 per mm²), yet stomatal density was found to be about half of that of leaves. Anatomical differences observed between petioles and pedicels were only minor. Stems of D. caryophyllus as well as petioles and pedicels of Z. aethiopica have a chlorenchyma-type tissue whose fine structure is quite similar to the leaf palisade chlorenchyma. Yet, the palisade of Z. aethiopica petioles and pedicels shows a peculiar arrangement: palisade cells are arranged with their long axis parallel to the longitudinal organ axis. Palisade tissue found in the aforementioned non-foliar plant organs in both species shows strong red chlorophyll auto-fluorescence under epi-fluorescence optics, consists of cells with abundant chloroplasts, possesses high percentage of intercellular spaces (13 and 20%, respectively) and its cells expose considerable part of their surface to the intercellular air. The fine structure of this stem palisade tissue along with the abundance of functional stomata found on the epidermis may support efficient photosynthesis.     

Overexpressing components of the nuclear transport apparatus causes severe growth symptoms in tobacco leaves

Regulating nucleo-cytoplasmic transport of RNA and protein is a key cellular control point. Perturbing the function of plant nuclear transport components can cause significant developmental defects and in this report we add an important line to this evidence. Overexpression of AtRAN1 or AtNUP62 in Nicotiana benthamiana causes significant damage to leaf tissue. This demonstrates that the precise control of nuclear transport is an important aspect of maintaining tissue integrity.