A Defense Pathway Linking Plasma Membrane and Chloroplasts and Co-opted by Pathogens.

Chloroplasts are crucial players in the activation of defensive hormonal responses during plant-pathogen interactions. Here, we show that a plant virus-encoded protein re-localizes from the plasma membrane to chloroplasts upon activation of plant defense, interfering with the chloroplast-dependent anti-viral salicylic acid (SA) biosynthesis. Strikingly, we have found that plant pathogens from different kingdoms seem to have convergently evolved to target chloroplasts and impair SA-dependent defenses following an association with membranes, which relies on the co-existence of two subcellular targeting signals, an N-myristoylation site and a chloroplast transit peptide. This pattern is also present in plant proteins, at least one of which conversely activates SA defenses from the chloroplast. Taken together, our results suggest that a pathway linking plasma membrane to chloroplasts and activating defense exists in plants and that such pathway has been co-opted by plant pathogens during host-pathogen co-evolution to promote virulence through suppression of SA responses.

The m6A reader ECT1 drives mRNA sequestration to dampen salicylic acid-dependent stress responses in Arabidopsis.

N 6-methyladenosine (m6A) is a common epitranscriptional mRNA modification in eukaryotes. Thirteen putative m6A readers, mostly annotated as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) proteins, have been identified in Arabidopsis (Arabidopsis thaliana), but few have been characterized. Here, we show that the Arabidopsis m6A reader ECT1 modulates salicylic acid (SA)-mediated plant stress responses. ECT1 undergoes liquid-liquid phase separation in vitro, and its N-terminal prion-like domain is critical for forming in vivo cytosolic biomolecular condensates in response to SA or bacterial pathogens. Fluorescence-activated particle sorting coupled with quantitative PCR analyses unveiled that ECT1 sequesters SA-induced m6A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses. Consistent with this finding, ECT1 overexpression promotes bacterial multiplication in plants. Collectively, our findings unequivocally link ECT1-associated cytosolic condensates to SA-dependent plant stress responses, advancing the current understanding of m6A readers and the SA signaling network.

FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

Combinatorial interactions between viral proteins expand the potential functional landscape of the tomato yellow leaf curl virus proteome.

Viruses manipulate the cells they infect in order to replicate and spread. Due to strict size restrictions, viral genomes have reduced genetic space; how the action of the limited number of viral proteins results in the cell reprogramming observed during the infection is a long-standing question. Here, we explore the hypothesis that combinatorial interactions may expand the functional landscape of the viral proteome. We show that the proteins encoded by a plant-infecting DNA virus, the geminivirus tomato yellow leaf curl virus (TYLCV), physically associate with one another in an intricate network, as detected by a number of protein-protein interaction techniques. Importantly, our results indicate that intra-viral protein-protein interactions can modify the subcellular localization of the proteins involved. Using one particular pairwise interaction, that between the virus-encoded C2 and CP proteins, as proof-of-concept, we demonstrate that the combination of viral proteins leads to novel transcriptional effects on the host cell. Taken together, our results underscore the importance of studying viral protein function in the context of the infection. We propose a model in which viral proteins might have evolved to extensively interact with other elements within the viral proteome, enlarging the potential functional landscape available to the pathogen.

Hierarchical regulatory module GENOMES UNCOUPLED1-GOLDEN2-LIKE1/2-WRKY18/40 modulates salicylic acid signaling.

Chloroplast-to-nucleus retrograde signaling (RS) pathways are critical in modulating plant development and stress adaptation. Among chloroplast proteins mediating RS pathways, GENOMES UNCOUPLED1 (GUN1) represses the transcription of the nuclear transcription factors GOLDEN2-LIKE1 (GLK1) and GLK2 that positively regulate chloroplast biogenesis. Given the extensive exploration of the function of GUN1 in biogenic RS carried out in previous years, our understanding of its role in plant stress responses remains scarce. Here, we revealed that GUN1 contributes to the expression of salicylic acid (SA)-responsive genes (SARGs) through transcriptional repression of GLK1/2 in Arabidopsis (Arabidopsis thaliana). Loss of GUN1 significantly compromised the SA responsiveness in plants, concomitant with the upregulation of GLK1/2 transcripts. In contrast, knockout of GLK1/2 potentiated the expression of SARGs and led to enhanced stress responses. Chromatin immunoprecipitation, coupled with quantitative PCR and related reverse genetic approaches, unveiled that in gun1, GLK1/2 might modulate SA-triggered stress responses by stimulating the expression of WRKY18 and WRKY40, transcriptional repressors of SARGs. In summary, we demonstrate that a hierarchical regulatory module, consisting of GUN1-GLK1/2-WRKY18/40, modulates SA signaling, opening a research avenue regarding a latent GUN1 function in plant-environment interactions.

Small but mighty: Functional landscape of the versatile geminivirus-encoded C4 protein.

The fast-paced evolution of viruses enables them to quickly adapt to the organisms they infect by constantly exploring the potential functional landscape of the proteins encoded in their genomes. Geminiviruses, DNA viruses infecting plants and causing devastating crop diseases worldwide, produce a limited number of multifunctional proteins that mediate the manipulation of the cellular environment to the virus' advantage. Among the proteins produced by the members of this family, C4, the smallest one described to date, is emerging as a powerful viral effector with unexpected versatility. C4 is the only geminiviral protein consistently subjected to positive selection and displays a number of dynamic subcellular localizations, interacting partners, and functions, which can vary between viral species. In this review, we aim to summarize our current knowledge on this remarkable viral protein, encompassing the different aspects of its multilayered diversity, and discuss what it can teach us about geminivirus evolution, invasion requirements, and virulence strategies.

PLANT NATRIURETIC PEPTIDE A and Its Putative Receptor PNP-R2 Antagonize Salicylic Acid-Mediated Signaling and Cell Death.

The plant stress hormone salicylic acid (SA) participates in local and systemic acquired resistance, which eventually leads to whole-plant resistance to bacterial pathogens. However, if SA-mediated signaling is not appropriately controlled, plants incur defense-associated fitness costs such as growth inhibition and cell death. Despite its importance, to date only a few components counteracting the SA-primed stress responses have been identified in Arabidopsis (Arabidopsis thaliana). These include other plant hormones such as jasmonic acid and abscisic acid, and proteins such as LESION SIMULATING DISEASE1, a transcription coregulator. Here, we describe PLANT NATRIURETIC PEPTIDE A (PNP-A), a functional analog to vertebrate atrial natriuretic peptides, that appears to antagonize the SA-mediated plant stress responses. While loss of PNP-A potentiates SA-mediated signaling, exogenous application of synthetic PNP-A or overexpression of PNP-A significantly compromises the SA-primed immune responses. Moreover, we identify a plasma membrane-localized receptor-like protein, PNP-R2, that interacts with PNP-A and is required to initiate the PNP-A-mediated intracellular signaling. In summary, our work identifies a peptide and its putative cognate receptor as counteracting both SA-mediated signaling and SA-primed cell death in Arabidopsis.

A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi.

RNA interference (RNAi) in plants can move from cell to cell, allowing for systemic spread of an antiviral immune response. How this cell-to-cell spread of silencing is regulated is currently unknown. Here, we describe that the C4 protein from Tomato yellow leaf curl virus can inhibit the intercellular spread of RNAi. Using this viral protein as a probe, we have identified the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1) as a positive regulator of the cell-to-cell movement of RNAi, and determined that BAM1 and its closest homolog, BAM2, play a redundant role in this process. C4 interacts with the intracellular domain of BAM1 and BAM2 at the plasma membrane and plasmodesmata, the cytoplasmic connections between plant cells, interfering with the function of these RLKs in the cell-to-cell spread of RNAi. Our results identify BAM1 as an element required for the cell-to-cell spread of RNAi and highlight that signaling components have been coopted to play multiple functions in plants.

An atypical HLH transcriptional regulator plays a novel and important role in strawberry ripened receptacle.

BACKGROUND: In soft fruits, the differential expression of many genes during development and ripening is responsible for changing their organoleptic properties. In strawberry fruit, although some genes involved in the metabolic regulation of the ripening process have been functionally characterized, some of the most studied genes correspond to transcription factors. High throughput transcriptomics analyses performed in strawberry red receptacle (Fragaria x ananassa) allowed us to identify a ripening-related gene that codes an atypical HLH (FaPRE1) with high sequence homology with the PACLOBUTRAZOL RESISTANCE (PRE) genes. PRE genes are atypical bHLH proteins characterized by the lack of a DNA-binding domain and whose function has been linked to the regulation of cell elongation processes. RESULTS: FaPRE1 sequence analysis indicates that this gene belongs to the subfamily of atypical bHLHs that also includes ILI-1 from rice, SlPRE2 from tomato and AtPRE1 from Arabidopsis, which are involved in transcriptional regulatory processes as repressors, through the blockage by heterodimerization of bHLH transcription factors. FaPRE1 presented a transcriptional model characteristic of a ripening-related gene with receptacle-specific expression, being repressed by auxins and activated by abscisic acid (ABA). However, its expression was not affected by gibberellic acid (GA3). On the other hand, the transitory silencing of FaPRE1 transcription by agroinfiltration in receptacle produced the down-regulation of a group of genes related to the ripening process while inducing the transcription of genes involved in receptacle growth and development. CONCLUSIONS: In summary, this work presents for the first time experimental data that support an important novel function for the atypical HLH FaPRE1 during the strawberry fruit ripening. We hypothesize that FaPRE1 modulates antagonistically the transcription of genes related to both receptacle growth and ripening. Thus, FaPRE1 would repress the expression of receptacle growth promoting genes in the ripened receptacle, while it would activate the expression of those genes related to the receptacle ripening process.

The fruit-specific transcription factor FaDOF2 regulates the production of eugenol in ripe fruit receptacles.

Only a few transcription factors have been described in the regulation of the strawberry (Fragaria x ananassa) fruit ripening process. Using a transcriptomic approach, we identified and functionally characterized FaDOF2, a DOF-type ripening-related transcription factor, which is hormonally regulated and specific to the receptacle, though high expression levels were also found in petals. The expression pattern of FaDOF2 correlated with eugenol content, a phenylpropanoid volatile, in both fruit receptacles and petals. When FaDOF2 expression was silenced in ripe strawberry receptacles, the expression of FaEOBII and FaEGS2, two key genes involved in eugenol production, were down-regulated. These fruits showed a concomitant decrease in eugenol content, which confirmed that FaDOF2 is a transcription factor that is involved in eugenol production in ripe fruit receptacles. By using the yeast two-hybrid system and bimolecular fluorescence complementation, we demonstrated that FaDOF2 interacts with FaEOBII, a previously reported regulator of eugenol production, which determines fine-tuning of the expression of key genes that are involved in eugenol production. These results provide evidence that FaDOF2 plays a subsidiary regulatory role with FaEOBII in the expression of genes encoding enzymes that control eugenol production. Taken together, our results provide new insights into the regulation of the volatile phenylpropanoid pathway in ripe strawberry receptacles.

Extensive transcriptomic studies on the roles played by abscisic acid and auxins in the development and ripening of strawberry fruits.

Strawberry is an ideal model for studying the molecular biology of the development and ripening of non-climacteric fruits. Hormonal regulation of gene expression along all these processes in strawberries is still to be fully elucidated. Although auxins and ABA have been pointed out as the major regulatory hormones, few high-throughput analyses have been carried out to date. The role for ethylene and gibberellins as regulatory hormones during the development and ripening of the strawberry fruit remain still elusive. By using a custom-made and high-quality oligo microarray platform done with over 32,000 probes including all of the genes actually described in the strawberry genome, we have analysed the expression of genes during the development and ripening in the receptacles of these fruits. We classify these genes into two major groups depending upon their temporal and developmental expression. First group are genes induced during the initial development stages. The second group encompasses genes induced during the final maturation and ripening processes. Each of these two groups has been also divided into four sub-groups according their pattern of hormonal regulation. By analyzing gene expression, we clearly show that auxins and ABA are the main and key hormones that combined or independently are responsible of the development and ripening process. Auxins are responsible for the receptacle fruit development and, at the same time¸ prevent ripening by repressing crucial genes. ABA regulates the expression of the vast majority of genes involved in the ripening. The main genes expressed under the control of these hormones are presented and their physiological rule discussed. We also conclude that ethylene and gibberellins do not seem to play a prominent role during these processes.

An R2R3-MYB Transcription Factor Regulates Eugenol Production in Ripe Strawberry Fruit Receptacles.

Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. In ripe strawberry (Fragaria × ananassa) fruit receptacles, eugenol is biosynthesized by eugenol synthase (FaEGS2). However, the transcriptional regulation of this process is still unknown. We have identified and functionally characterized an R2R3 MYB transcription factor (emission of benzenoid II [FaEOBII]) that seems to be the orthologous gene of PhEOBII from Petunia hybrida, which contributes to the regulation of eugenol biosynthesis in petals. The expression of FaEOBII was ripening related and fruit receptacle specific, although high expression values were also found in petals. This expression pattern of FaEOBII correlated with eugenol content in both fruit receptacle and petals. The expression of FaEOBII was repressed by auxins and activated by abscisic acid, in parallel to the ripening process. In ripe strawberry receptacles, where the expression of FaEOBII was silenced, the expression of cinnamyl alcohol dehydrogenase1 and FaEGS2, two structural genes involved in eugenol production, was down-regulated. A subsequent decrease in eugenol content in ripe receptacles was also observed, confirming the involvement of FaEOBII in eugenol metabolism. Additionally, the expression of FaEOBII was under the control of FaMYB10, another R2R3 MYB transcription factor that regulates the early and late biosynthetic genes from the flavonoid/phenylpropanoid pathway. In parallel, the amount of eugenol in FaMYB10-silenced receptacles was also diminished. Taken together, these data indicate that FaEOBII plays a regulating role in the volatile phenylpropanoid pathway gene expression that gives rise to eugenol production in ripe strawberry receptacles.

MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of Fragaria x ananassa fruits.

This work characterized the role of the R2R3-MYB10 transcription factor (TF) in strawberry fruit ripening. The expression of this TF takes place mainly in the fruit receptacle and is repressed by auxins and activated by abscisic acid (ABA), in parallel to the ripening process. Anthocyanin was not produced when FaMYB10 expression was transiently silenced in fruit receptacles. An increase in FaMYB10 expression was observed in water-stressed fruits, which was accompanied by an increase in both ABA and anthocyanin content. High-throughput transcriptomic analyses performed in fruits with downregulated FaMYB10 expression indicated that this TF regulates the expression of most of the Early-regulated Biosynthesis Genes (EBGs) and the Late-regulated Biosynthesis Genes (LBGs) genes involved in anthocyanin production in ripened fruit receptacles. Besides, the expression of FaMYB10 was not regulated by FaMYB1 and vice versa. Taken together, all these data clearly indicate that the Fragaria × ananassa MYB10 TF plays a general regulatory role in the flavonoid/phenylpropanoid pathway during the ripening of strawberry.

FaGAST2, a strawberry ripening-related gene, acts together with FaGAST1 to determine cell size of the fruit receptacle.

Numerous GAST-like genes have been reported in higher plants, but only one GAST-like gene (FaGAST1) has been described in strawberry so far. Herein, we have identified a novel strawberry FaGAST gene (FaGAST2) whose expression showed an increase throughout fruit receptacle development and ripening, coinciding with those stages where a decrease in fruit expansion processes (G3-W and R-OR stages) occurs. FaGAST2 only shares 31% and 15.7% amino acid and nucleotide sequence homology, respectively, with the previously reported FaGAST1 gene, but both genes contain a signal peptide and a highly conserved GASA domain (cysteine-rich domain) in the C-terminal region. FaGAST2 expression is mainly confined to the fruit receptacle and is not regulated by auxins, GA(3) or ABA, but is regulated by ethephon, an intracellular generator of ethylene. In addition, the expression of the FaGAST2 gene also increased under oxidative stress conditions (H(2)O(2) or Colletotrichum acutatum infection), suggesting a direct role for FaGAST2 protein in reactive oxygen species scavenging during fruit growth and ripening and during fungal infection. On the other hand, the overexpression of the FaGAST2 gene in different transgenic lines analyzed caused a delay in the growth of strawberry plants and a reduction in the size of the transgenic fruits. The histological studies performed in these fruits showed that their parenchymal cells were smaller than those of the controls, supporting a relationship between FaGAST2 gene expression, strawberry fruit cell elongation and fruit size. However, transitory silencing of FaGAST2 gene expression through RNA interference approaches revealed an increase in FaGAST1 expression, but no changes in fruit cell size were observed. These results support the hypothesis that both genes must act synergistically to determine fruit cell size during fruit development and ripening.

The strawberry (Fragariaxananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae.

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

The fruit ripening-related gene FaAAT2 encodes an acyl transferase involved in strawberry aroma biogenesis.

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.

Plasma membrane-to-organelle communication in plant stress signaling.

Intracellular compartments engage in extensive communication with one another, an essential ability for cells to respond and adapt to changing environmental and developmental conditions. The plasma membrane (PM), as the interface between the cellular and the outside media, plays a central role in the perception and relay of information about external stimuli, which needs to be ultimately addressed to the relevant subcellular organelles. Interest in PM-organelle communication has increased dramatically in recent years, as examples arise that illustrate different strategies through which information from the PM can be transmitted. In this review, we will discuss mechanisms enabling PM-to-organelle communication in plants, specifically in biotic and abiotic stress signaling.

The V2 Protein from the Geminivirus Tomato Yellow Leaf Curl Virus Largely Associates to the Endoplasmic Reticulum and Promotes the Accumulation of the Viral C4 Protein in a Silencing Suppression-Independent Manner.

Viruses are strict intracellular parasites that rely on the proteins encoded in their genomes for the effective manipulation of the infected cell that ultimately enables a successful infection. Viral proteins have to be produced during the cell invasion and takeover in sufficient amounts and in a timely manner. Silencing suppressor proteins evolved by plant viruses can boost the production of viral proteins; although, additional mechanisms for the regulation of viral protein production likely exist. The strongest silencing suppressor encoded by the geminivirus tomato yellow leaf curl virus (TYLCV) is V2: V2 suppresses both post-transcriptional and transcriptional gene silencing (PTGS and TGS), activities that are associated with its localization in punctate cytoplasmic structures and in the nucleus, respectively. However, V2 has been previously described to largely localize in the endoplasmic reticulum (ER), although the biological relevance of this distribution remains mysterious. Here, we confirm the association of V2 to the ER in Nicotiana benthamiana and assess the silencing suppression activity-independent impact of V2 on protein accumulation. Our results indicate that V2 has no obvious influence on the localization of ER-synthesized receptor-like kinases (RLKs) or ER quality control (ERQC)/ER-associated degradation (ERAD), but dramatically enhances the accumulation of the viral C4 protein, which is co-translationally myristoylated, possibly in proximity to the ER. By using the previously described V2C84S/86S mutant, in which the silencing suppression activity is abolished, we uncouple RNA silencing from the observed effect. Therefore, this work uncovers a novel function of V2, independent of its capacity to suppress silencing, in the promotion of the accumulation of another crucial viral protein.

Protocol for evaluating protein relocalization from the plasma membrane to chloroplasts.

We present a protocol for analyzing and evaluating the relocalization of proteins from the plasma membrane to chloroplasts. Some plant membrane-bound proteins carry dual targeting signals, e.g., a membrane-anchoring N-myristoylation motif and a chloroplast transit peptide. These proteins are predominantly targeted to membranes; upon certain cues, however, they can undergo detachment from membranes and relocalization to chloroplasts. This protocol combines imaging and biochemical analyses to track in a reliable and quantitative manner the relocalization of proteins between subcellular organelles. For complete details on the use and execution of this protocol, please refer to Medina-Puche et al. (2020).