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1.  Arabidopsis LIP5, a Positive Regulator of Multivesicular Body Biogenesis, Is a Critical Target of Pathogen-Responsive MAPK Cascade in Plant Basal Defense 
PLoS Pathogens  2014;10(7):e1004243.
Multivesicular bodies (MVBs) play essential roles in many cellular processes. The MVB pathway requires reversible membrane association of the endosomal sorting complexes required for transports (ESCRTs) for sustained protein trafficking. Membrane dissociation of ESCRTs is catalyzed by the AAA ATPase SKD1, which is stimulated by LYST-INTERACTING PROTEIN 5 (LIP5). We report here that LIP5 is a target of pathogen-responsive mitogen-activated protein kinases (MPKs) and plays a critical role in plant basal resistance. Arabidopsis LIP5 interacts with MPK6 and MPK3 and is phosphorylated in vitro by activated MPK3 and MPK6 and in vivo upon expression of MPK3/6-activating NtMEK2DD and pathogen infection. Disruption of LIP5 has little effects on flg22-, salicylic acid-induced defense responses but compromises basal resistance to Pseudomonas syringae. The critical role of LIP5 in plant basal resistance is dependent on its ability to interact with SKD1. Mutation of MPK phosphorylation sites in LIP5 does not affect interaction with SKD1 but reduces the stability and compromises the ability to complement the lip5 mutant phenotypes. Using the membrane-selective FM1–43 dye and transmission electron microscopy, we demonstrated that pathogen infection increases formation of both intracellular MVBs and exosome-like paramural vesicles situated between the plasma membrane and the cell wall in a largely LIP5-dependent manner. These results indicate that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system likely through relocalization of defense-related molecules.
Author Summary
Pathogen- and stress-responsive mitogen-activated protein kinases 3 and 6 (MPK3/6) cascade plays an important role in plant basal resistance to microbial pathogens. Here we showed that Arabidopsis MPK3 and MPK6 interact with and phosphorylate the LIP5 positive regulator of biogenesis of multivesicular bodies (MVBs), which are unique organelles containing small vesicles in their lumen. Disruption of LIP5 causes increased susceptibility to the bacterial pathogen Pseudomonas syringae. Compromised disease resistance of the lip5 mutants is associated with competent flg22- and salicylic acid-induced defense responses but compromised accumulation of intracellular MVBs and exosome-like paramural vesicles, which have previously been shown to be involved in the relocalization of defense-related molecules. Phosphorylation by MPK3/6 increases LIP5 stability, which is necessary for pathogen-induced MVB trafficking and basal disease resistance. Based on these results we conclude that the MVB pathway is positively regulated by pathogen-responsive MPK3/6 through LIP5 phosphorylation and plays a critical role in plant immune system probably through involvement in the relocalization of defense-related molecules.
doi:10.1371/journal.ppat.1004243
PMCID: PMC4092137  PMID: 25010425
2.  H2O2 mediates the crosstalk of brassinosteroid and abscisic acid in tomato responses to heat and oxidative stresses 
Journal of Experimental Botany  2014;65(15):4371-4383.
Summary
Brassinosteroids induce H2O2 accumulation from RBOH1-NADPH oxidase, which first induces ABA biosynthesis and stress tolerance, in turn leading to prolonged H2O2 production in both apoplast and chloroplast and stress tolerances.
The production of H2O2 is critical for brassinosteroid (BR)- and abscisic acid (ABA)-induced stress tolerance in plants. In this study, the relationship between BR and ABA in the induction of H2O2 production and their roles in response to heat and paraquat (PQ) oxidative stresses were studied in tomato. Both BR and ABA induced increases in RBOH1 gene expression, NADPH oxidase activity, apoplastic H2O2 accumulation, and heat and PQ stress tolerance in wild-type plants. BR could only induced transient increases in these responses in the ABA biosynthetic mutant notabilis (not), whereas ABA induced strong and prolonged increases in these responses in the BR biosynthetic mutant d ^im compared with wild-type plants. ABA levels were reduced in the BR biosynthetic mutant but could be elevated by exogenous BR. Silencing of RBOH1 compromised BR-induced apoplastic H2O2 production, ABA accumulation, and PQ stress responses; however, ABA-induced PQ stress responses were largely unchanged in the RBOH1-silenced plants. BR induces stress tolerance involving a positive feedback mechanism in which BR induces a rapid and transient H2O2 production by NADPH oxidase. The process in turn triggers increased ABA biosynthesis, leading to further increases in H2O2 production and prolonged stress tolerance. ABA induces H2O2 production in both the apoplastic and chloroplastic compartments.
doi:10.1093/jxb/eru217
PMCID: PMC4112640  PMID: 24899077
Abscisic acid; brassinosteroid; hydrogen peroxide; NADPH oxidase; Solanum lycopersicum; VIGS.
3.  E3 Ubiquitin Ligase CHIP and NBR1-Mediated Selective Autophagy Protect Additively against Proteotoxicity in Plant Stress Responses 
PLoS Genetics  2014;10(1):e1004116.
Plant stress responses require both protective measures that reduce or restore stress-inflicted damage to cellular structures and mechanisms that efficiently remove damaged and toxic macromolecules, such as misfolded and damaged proteins. We have recently reported that NBR1, the first identified plant autophagy adaptor with a ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting stress-induced, ubiquitinated protein aggregates for degradation by autophagy. Here we report a comprehensive genetic analysis of CHIP, a chaperone-associated E3 ubiquitin ligase from Arabidopsis thaliana implicated in mediating degradation of nonnative proteins by 26S proteasomes. We isolated two chip knockout mutants and discovered that they had the same phenotypes as the nbr1 mutants with compromised tolerance to heat, oxidative and salt stresses and increased accumulation of insoluble proteins under heat stress. To determine their functional interactions, we generated chip nbr1 double mutants and found them to be further compromised in stress tolerance and in clearance of stress-induced protein aggregates, indicating additive roles of CHIP and NBR1. Furthermore, stress-induced protein aggregates were still ubiquitinated in the chip mutants. Through proteomic profiling, we systemically identified heat-induced protein aggregates in the chip and nbr1 single and double mutants. These experiments revealed that highly aggregate-prone proteins such as Rubisco activase and catalases preferentially accumulated in the nbr1 mutant while a number of light-harvesting complex proteins accumulated at high levels in the chip mutant after a relatively short period of heat stress. With extended heat stress, aggregates for a large number of intracellular proteins accumulated in both chip and nbr1 mutants and, to a greater extent, in the chip nbr1 double mutant. Based on these results, we propose that CHIP and NBR1 mediate two distinct but complementary anti-proteotoxic pathways and protein's propensity to aggregate under stress conditions is one of the critical factors for pathway selection of protein degradation.
Author Summary
Environmental stresses such as heat cause generation of misfolded and damaged proteins, which are highly toxic and must be efficiently removed. In plants, NBR1, the first isolated autophagy receptor with an ubiquitin-association domain, plays a critical role in plant stress tolerance by targeting ubiquitinated protein aggregates under stress conditions for degradation by autophagy. To study how stress-induced misfolded and damaged proteins are detected and ubiquitinated in plant cells, we analyzed the chaperone-associated E3 ubiquitin ligase CHIP from Arabidopsis thaliana for its role in protection against proteotoxicity in plant stress responses. Disruption of Arabidopsis CHIP caused increased sensitivity to a spectrum of abiotic stresses as found in the Arabidopsis nbr1 mutants. Disruption of both Arabidopsis CHIP and NBR1 further compromised plant stress tolerance, indicating that their roles are additive. Furthermore, in the chip nbr1 double mutant, compromised heat tolerance was associated with increased accumulation of insoluble proteins derived mostly from heat-sensitive but biologically important proteins such as Rubisco activase, catalases and proteins required for protein synthesis and folding. Importantly, stress-induced protein aggregates were still highly ubiquitinated in the chip mutants. These results strongly suggest that CHIP and NBR1 function in two distinct but complementary anti-proteotoxic pathways in plant stress responses.
doi:10.1371/journal.pgen.1004116
PMCID: PMC3907298  PMID: 24497840
4.  Role and regulation of autophagy in heat stress responses of tomato plants 
As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates.
doi:10.3389/fpls.2014.00174
PMCID: PMC4012191  PMID: 24817875
autophagy; heat tolerance; tomato; WRKY33; NBR1; ATG5; ATG7
5.  RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato 
Journal of Experimental Botany  2013;65(2):595-607.
H2O2 and mitogen-activated protein kinase (MAPK) cascades play important functions in plant stress responses, but their roles in acclimation response remain unclear. This study examined the functions of H2O2 and MPK1/2 in acclimation-induced cross-tolerance in tomato plants. Mild cold, paraquat, and drought as acclimation stimuli enhanced tolerance to more severe subsequent chilling, photooxidative, and drought stresses. Acclimation-induced cross-tolerance was associated with increased transcript levels of RBOH1 and stress- and defence-related genes, elevated apoplastic H2O2 accumulation, increased activity of NADPH oxidase and antioxidant enzymes, reduced glutathione redox state, and activation of MPK1/2 in tomato. Virus-induced gene silencing of RBOH1, MPK1, and MPK2 or MPK1/2 all compromised acclimation-induced cross-tolerance and associated stress responses. Taken together, these results strongly suggest that acclimation-induced cross-tolerance is largely attributed to RBOH1-dependent H2O2 production at the apoplast, which may subsequently activate MPK1/2 to induce stress responses.
doi:10.1093/jxb/ert404
PMCID: PMC3904713  PMID: 24323505
Cross-tolerance; hydrogen peroxide; mitogen-activated protein kinase; reactive oxygen species; Respiratory burst oxidase homologue 1; signal transduction; Solanum lycopersicum.
6.  Brassinosteroids-Induced Systemic Stress Tolerance was Associated with Increased Transcripts of Several Defence-Related Genes in the Phloem in Cucumis sativus 
PLoS ONE  2013;8(6):e66582.
Brassinosteroids (BRs), a group of naturally occurring plant steroidal compounds, are essential for plant growth, development and stress tolerance. Recent studies showed that BRs could induce systemic tolerance to biotic and abiotic stresses; however, the molecular mechanisms by which BRs signals lead to responses in the whole plant are largely unknown. In this study, 24-epibrassinosteroid (EBR)-induced systemic tolerance in Cucumis sativus L. cv. Jinyan No. 4 was analyzed through the assessment of symptoms of photooxidative stress by chlorophyll fluorescence imaging pulse amplitude modulation. Expression of defense/stress related genes were induced in both treated local leaves and untreated systemic leaves by local EBR application. With the suppressive subtractive hybridization (SSH) library using cDNA from the phloem sap of EBR-treated plants as the tester and distilled water (DW)-treated plants as the driver, 14 transcripts out of 260 clones were identified. Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR) validated the specific up-regulation of these transcripts. Of the differentially expressed transcripts with known functions, transcripts for the selected four cDNAs, which encode an auxin-responsive protein (IAA14), a putative ankyrin-repeat protein, an F-box protein (PP2), and a major latex, pathogenesis-related (MLP)-like protein, were induced in local leaves, systemic leaves and roots after foliar application of EBR onto mature leaves. Our results demonstrated that EBR-induced systemic tolerance is accompanied with increased transcript of genes in the defense response in other organs. The potential role of phloem mRNAs as signaling components in mediating BR-regulated systemic resistance is discussed.
doi:10.1371/journal.pone.0066582
PMCID: PMC3686678  PMID: 23840504
7.  Guidelines for the use and interpretation of assays for monitoring autophagy 
Klionsky, Daniel J. | Abdalla, Fabio C. | Abeliovich, Hagai | Abraham, Robert T. | Acevedo-Arozena, Abraham | Adeli, Khosrow | Agholme, Lotta | Agnello, Maria | Agostinis, Patrizia | Aguirre-Ghiso, Julio A. | Ahn, Hyung Jun | Ait-Mohamed, Ouardia | Ait-Si-Ali, Slimane | Akematsu, Takahiko | Akira, Shizuo | Al-Younes, Hesham M. | Al-Zeer, Munir A. | Albert, Matthew L. | Albin, Roger L. | Alegre-Abarrategui, Javier | Aleo, Maria Francesca | Alirezaei, Mehrdad | Almasan, Alexandru | Almonte-Becerril, Maylin | Amano, Atsuo | Amaravadi, Ravi K. | Amarnath, Shoba | Amer, Amal O. | Andrieu-Abadie, Nathalie | Anantharam, Vellareddy | Ann, David K. | Anoopkumar-Dukie, Shailendra | Aoki, Hiroshi | Apostolova, Nadezda | Arancia, Giuseppe | Aris, John P. | Asanuma, Katsuhiko | Asare, Nana Y.O. | Ashida, Hisashi | Askanas, Valerie | Askew, David S. | Auberger, Patrick | Baba, Misuzu | Backues, Steven K. | Baehrecke, Eric H. | Bahr, Ben A. | Bai, Xue-Yuan | Bailly, Yannick | Baiocchi, Robert | Baldini, Giulia | Balduini, Walter | Ballabio, Andrea | Bamber, Bruce A. | Bampton, Edward T.W. | Juhász, Gábor | Bartholomew, Clinton R. | Bassham, Diane C. | Bast, Robert C. | Batoko, Henri | Bay, Boon-Huat | Beau, Isabelle | Béchet, Daniel M. | Begley, Thomas J. | Behl, Christian | Behrends, Christian | Bekri, Soumeya | Bellaire, Bryan | Bendall, Linda J. | Benetti, Luca | Berliocchi, Laura | Bernardi, Henri | Bernassola, Francesca | Besteiro, Sébastien | Bhatia-Kissova, Ingrid | Bi, Xiaoning | Biard-Piechaczyk, Martine | Blum, Janice S. | Boise, Lawrence H. | Bonaldo, Paolo | Boone, David L. | Bornhauser, Beat C. | Bortoluci, Karina R. | Bossis, Ioannis | Bost, Frédéric | Bourquin, Jean-Pierre | Boya, Patricia | Boyer-Guittaut, Michaël | Bozhkov, Peter V. | Brady, Nathan R | Brancolini, Claudio | Brech, Andreas | Brenman, Jay E. | Brennand, Ana | Bresnick, Emery H. | Brest, Patrick | Bridges, Dave | Bristol, Molly L. | Brookes, Paul S. | Brown, Eric J. | Brumell, John H. | Brunetti-Pierri, Nicola | Brunk, Ulf T. | Bulman, Dennis E. | Bultman, Scott J. | Bultynck, Geert | Burbulla, Lena F. | Bursch, Wilfried | Butchar, Jonathan P. | Buzgariu, Wanda | Bydlowski, Sergio P. | Cadwell, Ken | Cahová, Monika | Cai, Dongsheng | Cai, Jiyang | Cai, Qian | Calabretta, Bruno | Calvo-Garrido, Javier | Camougrand, Nadine | Campanella, Michelangelo | Campos-Salinas, Jenny | Candi, Eleonora | Cao, Lizhi | Caplan, Allan B. | Carding, Simon R. | Cardoso, Sandra M. | Carew, Jennifer S. | Carlin, Cathleen R. | Carmignac, Virginie | Carneiro, Leticia A.M. | Carra, Serena | Caruso, Rosario A. | Casari, Giorgio | Casas, Caty | Castino, Roberta | Cebollero, Eduardo | Cecconi, Francesco | Celli, Jean | Chaachouay, Hassan | Chae, Han-Jung | Chai, Chee-Yin | Chan, David C. | Chan, Edmond Y. | Chang, Raymond Chuen-Chung | Che, Chi-Ming | Chen, Ching-Chow | Chen, Guang-Chao | Chen, Guo-Qiang | Chen, Min | Chen, Quan | Chen, Steve S.-L. | Chen, WenLi | Chen, Xi | Chen, Xiangmei | Chen, Xiequn | Chen, Ye-Guang | Chen, Yingyu | Chen, Yongqiang | Chen, Yu-Jen | Chen, Zhixiang | Cheng, Alan | Cheng, Christopher H.K. | Cheng, Yan | Cheong, Heesun | Cheong, Jae-Ho | Cherry, Sara | Chess-Williams, Russ | Cheung, Zelda H. | Chevet, Eric | Chiang, Hui-Ling | Chiarelli, Roberto | Chiba, Tomoki | Chin, Lih-Shen | Chiou, Shih-Hwa | Chisari, Francis V. | Cho, Chi Hin | Cho, Dong-Hyung | Choi, Augustine M.K. | Choi, DooSeok | Choi, Kyeong Sook | Choi, Mary E. | Chouaib, Salem | Choubey, Divaker | Choubey, Vinay | Chu, Charleen T. | Chuang, Tsung-Hsien | Chueh, Sheau-Huei | Chun, Taehoon | Chwae, Yong-Joon | Chye, Mee-Len | Ciarcia, Roberto | Ciriolo, Maria R. | Clague, Michael J. | Clark, Robert S.B. | Clarke, Peter G.H. | Clarke, Robert | Codogno, Patrice | Coller, Hilary A. | Colombo, María I. | Comincini, Sergio | Condello, Maria | Condorelli, Fabrizio | Cookson, Mark R. | Coombs, Graham H. | Coppens, Isabelle | Corbalan, Ramon | Cossart, Pascale | Costelli, Paola | Costes, Safia | Coto-Montes, Ana | Couve, Eduardo | Coxon, Fraser P. | Cregg, James M. | Crespo, José L. | Cronjé, Marianne J. | Cuervo, Ana Maria | Cullen, Joseph J. | Czaja, Mark J. | D'Amelio, Marcello | Darfeuille-Michaud, Arlette | Davids, Lester M. | Davies, Faith E. | De Felici, Massimo | de Groot, John F. | de Haan, Cornelis A.M. | De Martino, Luisa | De Milito, Angelo | De Tata, Vincenzo | Debnath, Jayanta | Degterev, Alexei | Dehay, Benjamin | Delbridge, Lea M.D. | Demarchi, Francesca | Deng, Yi Zhen | Dengjel, Jörn | Dent, Paul | Denton, Donna | Deretic, Vojo | Desai, Shyamal D. | Devenish, Rodney J. | Di Gioacchino, Mario | Di Paolo, Gilbert | Di Pietro, Chiara | Díaz-Araya, Guillermo | Díaz-Laviada, Inés | Diaz-Meco, Maria T. | Diaz-Nido, Javier | Dikic, Ivan | Dinesh-Kumar, Savithramma P. | Ding, Wen-Xing | Distelhorst, Clark W. | Diwan, Abhinav | Djavaheri-Mergny, Mojgan | Dokudovskaya, Svetlana | Dong, Zheng | Dorsey, Frank C. | Dosenko, Victor | Dowling, James J. | Doxsey, Stephen | Dreux, Marlène | Drew, Mark E. | Duan, Qiuhong | Duchosal, Michel A. | Duff, Karen E. | Dugail, Isabelle | Durbeej, Madeleine | Duszenko, Michael | Edelstein, Charles L. | Edinger, Aimee L. | Egea, Gustavo | Eichinger, Ludwig | Eissa, N. Tony | Ekmekcioglu, Suhendan | El-Deiry, Wafik S. | Elazar, Zvulun | Elgendy, Mohamed | Ellerby, Lisa M. | Eng, Kai Er | Engelbrecht, Anna-Mart | Engelender, Simone | Erenpreisa, Jekaterina | Escalante, Ricardo | Esclatine, Audrey | Eskelinen, Eeva-Liisa | Espert, Lucile | Espina, Virginia | Fan, Huizhou | Fan, Jia | Fan, Qi-Wen | Fan, Zhen | Fang, Shengyun | Fang, Yongqi | Fanto, Manolis | Fanzani, Alessandro | Farkas, Thomas | Farre, Jean-Claude | Faure, Mathias | Fechheimer, Marcus | Feng, Carl G. | Feng, Jian | Feng, Qili | Feng, Youji | Fésüs, László | Feuer, Ralph | Figueiredo-Pereira, Maria E. | Fimia, Gian Maria | Fingar, Diane C. | Finkbeiner, Steven | Finkel, Toren | Finley, Kim D. | Fiorito, Filomena | Fisher, Edward A. | Fisher, Paul B. | Flajolet, Marc | Florez-McClure, Maria L. | Florio, Salvatore | Fon, Edward A. | Fornai, Francesco | Fortunato, Franco | Fotedar, Rati | Fowler, Daniel H. | Fox, Howard S. | Franco, Rodrigo | Frankel, Lisa B. | Fransen, Marc | Fuentes, José M. | Fueyo, Juan | Fujii, Jun | Fujisaki, Kozo | Fujita, Eriko | Fukuda, Mitsunori | Furukawa, Ruth H. | Gaestel, Matthias | Gailly, Philippe | Gajewska, Malgorzata | Galliot, Brigitte | Galy, Vincent | Ganesh, Subramaniam | Ganetzky, Barry | Ganley, Ian G. | Gao, Fen-Biao | Gao, George F. | Gao, Jinming | Garcia, Lorena | Garcia-Manero, Guillermo | Garcia-Marcos, Mikel | Garmyn, Marjan | Gartel, Andrei L. | Gatti, Evelina | Gautel, Mathias | Gawriluk, Thomas R. | Gegg, Matthew E. | Geng, Jiefei | Germain, Marc | Gestwicki, Jason E. | Gewirtz, David A. | Ghavami, Saeid | Ghosh, Pradipta | Giammarioli, Anna M. | Giatromanolaki, Alexandra N. | Gibson, Spencer B. | Gilkerson, Robert W. | Ginger, Michael L. | Ginsberg, Henry N. | Golab, Jakub | Goligorsky, Michael S. | Golstein, Pierre | Gomez-Manzano, Candelaria | Goncu, Ebru | Gongora, Céline | Gonzalez, Claudio D. | Gonzalez, Ramon | González-Estévez, Cristina | González-Polo, Rosa Ana | Gonzalez-Rey, Elena | Gorbunov, Nikolai V. | Gorski, Sharon | Goruppi, Sandro | Gottlieb, Roberta A. | Gozuacik, Devrim | Granato, Giovanna Elvira | Grant, Gary D. | Green, Kim N. | Gregorc, Ales | Gros, Frédéric | Grose, Charles | Grunt, Thomas W. | Gual, Philippe | Guan, Jun-Lin | Guan, Kun-Liang | Guichard, Sylvie M. | Gukovskaya, Anna S. | Gukovsky, Ilya | Gunst, Jan | Gustafsson, Åsa B. | Halayko, Andrew J. | Hale, Amber N. | Halonen, Sandra K. | Hamasaki, Maho | Han, Feng | Han, Ting | Hancock, Michael K. | Hansen, Malene | Harada, Hisashi | Harada, Masaru | Hardt, Stefan E. | Harper, J. 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Angus | Meijer, Alfred J. | Meisler, Miriam H. | Meléndez, Alicia | Melia, Thomas J. | Melino, Gerry | Mena, Maria A. | Menendez, Javier A. | Menna-Barreto, Rubem F. 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Ashutosh | Rathmell, Jeffrey C. | Ravikumar, Brinda | Ray, Swapan K. | Reed, Bruce H. | Reed, John C. | Reggiori, Fulvio | Régnier-Vigouroux, Anne | Reichert, Andreas S. | Reiners, John J. | Reiter, Russel J. | Ren, Jun | Revuelta, José L. | Rhodes, Christopher J. | Ritis, Konstantinos | Rizzo, Elizete | Robbins, Jeffrey | Roberge, Michel | Roca, Hernan | Roccheri, Maria C. | Rocchi, Stephane | Rodemann, H. Peter | Rodríguez de Córdoba, Santiago | Rohrer, Bärbel | Roninson, Igor B. | Rosen, Kirill | Rost-Roszkowska, Magdalena M. | Rouis, Mustapha | Rouschop, Kasper M.A. | Rovetta, Francesca | Rubin, Brian P. | Rubinsztein, David C. | Ruckdeschel, Klaus | Rucker, Edmund B. | Rudich, Assaf | Rudolf, Emil | Ruiz-Opazo, Nelson | Russo, Rossella | Rusten, Tor Erik | Ryan, Kevin M. | Ryter, Stefan W. | Sabatini, David M. | Sadoshima, Junichi | Saha, Tapas | Saitoh, Tatsuya | Sakagami, Hiroshi | Sakai, Yasuyoshi | Salekdeh, Ghasem Hoseini | Salomoni, Paolo | Salvaterra, Paul M. | Salvesen, Guy | Salvioli, Rosa | Sanchez, Anthony M.J. | Sánchez-Alcázar, José A. | Sánchez-Prieto, Ricardo | Sandri, Marco | Sankar, Uma | Sansanwal, Poonam | Santambrogio, Laura | Saran, Shweta | Sarkar, Sovan | Sarwal, Minnie | Sasakawa, Chihiro | Sasnauskiene, Ausra | Sass, Miklós | Sato, Ken | Sato, Miyuki | Schapira, Anthony H.V. | Scharl, Michael | Schätzl, Hermann M. | Scheper, Wiep | Schiaffino, Stefano | Schneider, Claudio | Schneider, Marion E. | Schneider-Stock, Regine | Schoenlein, Patricia V. | Schorderet, Daniel F. | Schüller, Christoph | Schwartz, Gary K. | Scorrano, Luca | Sealy, Linda | Seglen, Per O. | Segura-Aguilar, Juan | Seiliez, Iban | Seleverstov, Oleksandr | Sell, Christian | Seo, Jong Bok | Separovic, Duska | Setaluri, Vijayasaradhi | Setoguchi, Takao | Settembre, Carmine | Shacka, John J. | Shanmugam, Mala | Shapiro, Irving M. | Shaulian, Eitan | Shaw, Reuben J. | Shelhamer, James H. | Shen, Han-Ming | Shen, Wei-Chiang
Autophagy  2012;8(4):445-544.
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
doi:10.4161/auto.19496
PMCID: PMC3404883  PMID: 22966490
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
8.  NBR1-Mediated Selective Autophagy Targets Insoluble Ubiquitinated Protein Aggregates in Plant Stress Responses 
PLoS Genetics  2013;9(1):e1003196.
Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.
Author Summary
Autophagy is an evolutionarily conserved process that sequestrates and delivers cytoplasmic macromolecules and organelles to the vacuoles or lysosomes for degradation. In plants, autophagy is involved in supplying internal nutrients during starvation and in promoting cell survival during senescence and during biotic and abiotic stresses. Arabidopsis NBR1 is a homolog of mammalian autophagy cargo adaptors P62 and NBR1. Disruption of Arabidopsis NBR1 caused increased sensitivity to a spectrum of abiotic stresses but had no significant effect on plant senescence, responses to carbon starvation, or resistance to a necrotrophic pathogen. NBR1 contains an ubiquitin-binding domain, and the compromised stress tolerance of autophagy mutants was associated with increased accumulation of NBR1 and ubiquitin-positive cellular protein aggregates in the insoluble protein fraction under stress conditions. Based on these results, we propose that NBR1 targets ubiquitinated protein aggregates most likely derived from denatured and otherwise damaged nonnative proteins for autophagic clearance under stress conditions.
doi:10.1371/journal.pgen.1003196
PMCID: PMC3547818  PMID: 23341779
9.  Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress 
BMC Plant Biology  2010;10:281.
Background
WRKY transcription factors are involved in plant responses to both biotic and abiotic stresses. Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors interact both physically and functionally in plant defense responses. However, their role in plant abiotic stress response has not been directly analyzed.
Results
We report that the three WRKYs are involved in plant responses to abscisic acid (ABA) and abiotic stress. Through analysis of single, double, and triple mutants and overexpression lines for the WRKY genes, we have shown that WRKY18 and WRKY60 have a positive effect on plant ABA sensitivity for inhibition of seed germination and root growth. The same two WRKY genes also enhance plant sensitivity to salt and osmotic stress. WRKY40, on the other hand, antagonizes WRKY18 and WRKY60 in the effect on plant sensitivity to ABA and abiotic stress in germination and growth assays. Both WRKY18 and WRKY40 are rapidly induced by ABA, while induction of WRKY60 by ABA is delayed. ABA-inducible expression of WRKY60 is almost completely abolished in the wrky18 and wrky40 mutants. WRKY18 and WRKY40 recognize a cluster of W-box sequences in the WRKY60 promoter and activate WRKY60 expression in protoplasts. Thus, WRKY60 might be a direct target gene of WRKY18 and WRKY40 in ABA signaling. Using a stable transgenic reporter/effector system, we have shown that both WRKY18 and WRKY60 act as weak transcriptional activators while WRKY40 is a transcriptional repressor in plant cells.
Conclusions
We propose that the three related WRKY transcription factors form a highly interacting regulatory network that modulates gene expression in both plant defense and stress responses by acting as either transcription activator or repressor.
doi:10.1186/1471-2229-10-281
PMCID: PMC3023790  PMID: 21167067
10.  Biosynthesis of salicylic acid in plants 
Plant Signaling & Behavior  2009;4(6):493-496.
Salicylic acid (SA) is an important signal molecule in plants. Two pathways of SA biosynthesis have been proposed in plants. Biochemical studies using isotope feeding have suggested that plants synthesize SA from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL). Silencing of PAL genes in tobacco or chemical inhibition of PAL activity in Arabidopsis, cucumber and potato reduces pathogen-induced SA accumulation. Genetic studies, on the other hand, indicate that the bulk of SA is produced from isochorismate. In bacteria, SA is synthesized from chorismate through two reactions catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Arabidopsis contains two ICS genes but has no gene encoding proteins similar to the bacterial IPL. Thus, how SA is synthesized in plants is not fully elucidated. Two recently identified Arabidopsis genes, PBS3 and EPS1, are important for pathogen-induced SA accumulation. PBS3 encodes a member of the acyl-adenylate/thioester-forming enzyme family and EPS1 encodes a member of the BAHD acyltransferase superfamily. PBS3 and EPS1 may be directly involved in the synthesis of an important precursor or regulatory molecule for SA biosynthesis. The pathways and regulation of SA biosynthesis in plants may be more complicated than previously thought.
PMCID: PMC2688294  PMID: 19816125
salicylic acid biosynthesis; isochorismate synthase; phenylalanine ammonia lyase
11.  Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens 
BMC Plant Biology  2008;8:68.
Background
Plant WRKY DNA-binding transcription factors are involved in plant responses to biotic and abiotic responses. It has been previously shown that Arabidopsis WRKY3 and WRKY4, which encode two structurally similar WRKY transcription factors, are induced by pathogen infection and salicylic acid (SA). However, the role of the two WRKY transcription factors in plant disease resistance has not been directly analyzed.
Results
Both WRKY3 and WRKY4 are nuclear-localized and specifically recognize the TTGACC W-box sequences in vitro. Expression of WRKY3 and WRKY4 was induced rapidly by stress conditions generated by liquid infiltration or spraying. Stress-induced expression of WRKY4 was further elevated by pathogen infection and SA treatment. To determine directly their role in plant disease resistance, we have isolated T-DNA insertion mutants and generated transgenic overexpression lines for WRKY3 and WRKY4. Both the loss-of-function mutants and transgenic overexpression lines were examined for responses to the biotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. The wrky3 and wrky4 single and double mutants exhibited more severe disease symptoms and support higher fungal growth than wild-type plants after Botrytis infection. Although disruption of WRKY3 and WRKY4 did not have a major effect on plant response to P. syringae, overexpression of WRKY4 greatly enhanced plant susceptibility to the bacterial pathogen and suppressed pathogen-induced PR1 gene expression.
Conclusion
The nuclear localization and sequence-specific DNA-binding activity support that WRKY3 and WRKY4 function as transcription factors. Functional analysis based on T-DNA insertion mutants and transgenic overexpression lines indicates that WRKY3 and WRKY4 have a positive role in plant resistance to necrotrophic pathogens and WRKY4 has a negative effect on plant resistance to biotrophic pathogens.
doi:10.1186/1471-2229-8-68
PMCID: PMC2464603  PMID: 18570649
12.  Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae 
BMC Plant Biology  2007;7:2.
Background
A common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant WRKY transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific WRKY DNA-binding transcription factors in plant defense.
Results
We analyzed the role of the WRKY25 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. WRKY25 protein recognizes the TTGACC W-box sequences and its translational fusion with green fluorescent protein is localized to the nucleus. WRKY25 expression is responsive to general environmental stress. Analysis of stress-induced WRKY25 in the defense signaling mutants npr1, sid2, ein2 and coi1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway and negatively regulated by the jasmonic acid signaling pathway. Two independent T-DNA insertion mutants for WRKY25 supported normal growth of a virulent strain of P. syringae but developed reduced disease symptoms after infection. By contrast, Arabidopsis constitutively overexpressing WRKY25 supported enhanced growth of P. syringae and displayed increased disease symptom severity as compared to wild-type plants. These WRKY25-overexpressing plants also displayed reduced expression of the SA-regulated PR1 gene after the pathogen infection, despite normal levels of free SA.
Conclusion
The nuclear localization and sequence-specific DNA-binding activity support that WRKY25 functions as a transcription factor. Based on analysis of both T-DNA insertion mutants and transgenic overexpression lines, stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae. This proposed role is consistent with the recent finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses.
doi:10.1186/1471-2229-7-2
PMCID: PMC1780049  PMID: 17214894

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