Abstract Plant viruses have evolved diverse strategies to facilitate their movement and survival within the host. Among them, geminiviruses co-opt host cellular machinery to replicate and disseminate. Traditionally, viral propagation has been associated with intercellular symplastic trafficking mediated by plasmodesmata and viral movement proteins. However, recent evidence demonstrated that the plant RNA virus turnip mosaic virus (TuMV) components are associated with extracellular vesicles (EVs). EVs are membrane-bound structures secreted to the extracellular space to potentially mediate several plant-pathogen interactions such as cross-kingdom RNA interference or the delivery of stress response proteins. In animals, EVs facilitate viral transmission both within the host and across species, but knowledge about their potential roles in plant viral infection is scarce. In this study, we demonstrate that EVs isolated from geminivirus-infected plants contain complete viral genomes and both capsid and viral movement proteins. Furthermore, these EV fractions were demonstrated to be infectious when mechanically inoculated onto naïve plants. This discovery suggests that EVs may serve as alternative carriers for geminivirus components, enabling long-range transport or potentially modulating host immune responses, and highlights geminiviral capacity to transverse membrane boundaries, essential for circulative arbovirus propagation in their insect vectors. Significance Statement Viruses are obligate intracellular parasites that shape ecological communities and crucially challenge animal and plant health worldwide. Conversely to animal viruses, plant-infecting viruses rely on plasmodesmata to disseminate through their host and establish systemic infection. Nonetheless, most plant viruses are insect-transmitted whose ecological cycle relies on their insect vector spread. Thus, strategies to cross continuous membrane barriers are essential for their dissemination and may be potentially conserved in plant hosts. Our discovery that infectious viral entities are associated with EVs reveals an alternative pathway for geminiviral movement within plant hosts that could facilitate vector transmission, challenging our long-standing understanding of plant virus biology and expanding the current conception of plant viral pathology.

Plant viruses cause symptoms with devastating consequences for agriculture. However, the molecular mechanisms underlying symptom development in viral infections remain largely unexplored. Here, we show that tomato yellow leaf curl virus (TYLCV) interferes with host developmental programs through a host-mimicking domain present in the viral C4 protein. This domain mediates the interaction between C4 and a family of RCC1-like domain-containing (RLD) proteins, previously shown to be required for proper plant development and environmental responses. C4 outcompetes an endogenous interactor of RLDs, hijacking RLD proteins to the plasma membrane and disrupting their function in orchestrating endomembrane trafficking and polar auxin transport. Strikingly, macroscopic symptoms do not affect viral accumulation in the plant but serve as attractants for the insect vector, presumably promoting pathogen spread in an ecological context. Our work sheds light on the molecular underpinnings and biological relevance of symptom development triggered by TYLCV in tomato. Since most plant viruses are insect-transmitted, the principles described here might have broad applicability to crop-virus interactions.

Circular Rep-encoding single-stranded (CRESS)-DNA viruses comprise a diverse group of viruses that use rolling-circle replication to copy their genomes. They infect organisms in almost all branches of the eukaryotic tree of life. A hallmark of all CRESS-DNA viruses is the presence of a single conserved protein, the replication initiator protein (Rep), that orchestrates viral replication by exploiting the host DNA replication machinery. In the case of the plant-infecting Geminiviridae, this multifunctional protein both recruits the host DNA replication machinery and manipulates post-translational modification including small ubiquitin-like modifier (SUMO) conjugation. In fact, Rep from two different geminiviruses, tomato yellow leaf curl virus (TYLCV; Begomovirus coheni) and tomato golden mosaic virus (TGMV; B. solanum aureimusivi), was earlier shown to interact with the SUMO conjugating enzyme SCE1. Here, we demonstrate that TYLCV C1/Rep protein also interacts with Arabidopsis SUMO1 and identify a SUMO interacting motif (SIM) located in the C-terminal SF3 helicase/ATPase domain. Remarkably, a functional SIM proved to be important for the interaction of Rep with both SUMO1 and SCE1. The same motif in the Rep ORF was essential for TYLCV viral replication, disease symptom formation, and systemic movement from an infectious clone, and Rep ATPase activity. Together, our findings thus connect the interaction between Rep and the SUMO machinery with TYLCV viral replication.IMPORTANCEThe identification of a non-canonical SUMO-interacting motif (SIM) within the Rep protein of tomato yellow leaf curl virus (TYLCV) reveals a connection between viral replication and a protein modification, i.e., SUMOylation. Importantly, the motif is conserved between Rep proteins from different geminiviruses. Functionally, the motif was critical for Rep's interaction with components of the SUMO machinery, for viral DNA replication, and for its ATPase activity. In particular, the third position of the motif was important for each of these activities. We thus uncover a hitherto undescribed mechanism on how geminiviruses recruit the SUMO machinery.

The vesicle trafficking system enables multidirectional cargo fluxes between endomembrane compartments. However, vesicle trafficking plays dual roles during pathogen infections. In plants, it mediates autophagic immune responses but can also be hijacked by pathogens to facilitate successful infections. We demonstrate that vesicle trafficking machinery acts as a double-edged sword during infection by the geminivirus tomato yellow leaf curl Sardinia virus (TYLCSaV) in Nicotiana benthamiana. Virus-induced gene silencing of eight genes encoding key vesicle trafficking regulators revealed contrasting outcomes. Silencing of NbSAR1 and NbAP-1γ significantly increased systemic geminiviral DNA accumulation, whereas silencing of Nbδ-COP, NbARF1 and clathrin genes almost completely abolished infection. Notably, this inhibition is hypothesized to result from direct or indirect impairment in viral movement, as replication remained unaffected by gene silencing. The observed effects affect other geminiviruses, including tomato yellow leaf curl virus (TYLCV) and beet curly top virus (BCTV), but not unrelated pathogens, such as the RNA potato virus X (PVX) or the plant pathogenic bacterium Pseudomonas syringae. These findings suggest that while the vacuolar and autophagy branches of the vesicle trafficking system might mediate antiviral autophagic defence responses, the integrity of endocytosis and retrograde transport is essential for systemic geminiviral infection.

Viral replicons are valuable tools in plant biotechnology, widely utilized to increase recombinant protein production. Their ability to amplify gene dosage in a trigger-dependent manner also opens doors to regulatory applications. This work focuses on optimizing geminivirus-based vectors for Synthetic Biology applications in plants, using autobioluminescence as a sensitive, real-time reporter to characterize gene expression. Specifically, geminivirus-based synthetic replicons derived from bean yellow dwarf virus (BeYDV), tomato yellow leaf curl virus (TYLCV), and beet curly top virus (BCTV) were engineered and assessed for basal expression, inducibility, and recombinant protein coexpression potential. Our study provided insights into the strengths and limitations of each geminiviral replicon. BeYDV replicon displayed a robust activation profile suitable for complex tasks such as multigene expression, while TYLCV showed high expression levels despite moderate basal leakage. In contrast, BCTV demonstrated less favorable control and expression levels. Through a bioluminescence-based screening, the TYLCV system was further optimized to improve regulatory precision. These findings highlight the versatility of geminivirus replicons, paving the way for future engineering of synthetic gene circuits in plants.

Viruses comprise the most abundant genetic material in the biosphere; however, global viral genomic population (virome) has been largely underestimated. Recently, high-throughput sequencing (HTS) has provided a powerful tool for the detection of known viruses and the discovery of novel viral species from environmental and individual samples using metagenomics and ecogenomics approaches, respectively. Viruses with circular DNA single-stranded (ssDNA) genomes belonging to the begomovirus genera (family Geminiviridae) constitute the largest group of emerging plant viruses worldwide. The knowledge of begomoviruses viromes is mostly restricted to crop plant systems; nevertheless, it has been described that noncultivated plants specifically at the interface between wild and cultivated plants are important reservoirs leading to viral evolution and the emergence of new diseases. Here we present a protocol that allows the identification and isolation of known and novel begomoviruses species infecting cultivated and noncultivated plant species. The method consists of circular viral molecules enrichment by rolling circle amplification (RCA) from begomovirus-positive total plant DNA, followed by NGS-based metagenomic sequencing. Subsequently, metagenomic reads are processed for taxonomic classification using Viromescan software and a customized Geminiviridae family database, and begomovirus-related reads are used for contigs assembly and annotation using Spades software and Blastn algorithm, respectively. Then, the obtained begomovirus-related signatures are used as templates for specific primers design and implemented for PCR-based ecogenomic identification of individual samples harboring the corresponding viral species. Lastly, full-length begomovirus genomes are obtained by RCA-based amplification from total plant DNA of selected individual samples, cloning, and viral molecular identity corroborated by Sanger sequencing. Conclusively, the identification and isolation of a novel monopartite begomovirus species native to the New World (NW) named Gallium leaf deformation virus (GLDV) is shown.

CRESS-DNA viruses form a diverse group of viruses that use rolling-circle replication to replicate their genomes. They infect organisms in almost all branches of the eukaryotic tree of life. All CRESS-DNA viruses have one protein in common, the Replication initiator protein (Rep), which orchestrates viral replication using the host DNA replication machinery. In the case of the plant-infecting Geminiviridae , this multifunctional protein both recruits the host DNA replication machinery and manipulates posttranslational modification including Small ubiquitin-like modifier (SUMO) conjugation. In fact, Rep from two different geminiviruses, Tomato yellow leaf curl virus (TYLCV) and Tomato golden mosaic virus (TGMV), was shown to interact with the SUMO conjugating enzyme SCE1. Here, we demonstrate that also TYLCV Rep interacts with Arabidopsis SUMO1 and report on a SUMO interacting motif (SIM) in the SF3 helicase domain of Rep. Remarkably, an intact SIM proved to be important for the interaction of Rep with both SUMO1 and SCE1. The same motif was also essential for viral replication and Rep ATPase activity. Our findings thus connect the interaction between Rep and the SUMO machinery with viral replication of TYLCV. Importance The identification of a non-canonical SUMO-interacting motif (SIM) within the Rep protein of Tomato yellow leaf curl virus (TYLCV) reveals a connection between viral replication and a protein modification, SUMOylation. Importantly, the motif was found to be conserved between Rep proteins from different geminiviruses. Functionally, the motif was critical for the interaction of Rep with proteins of the SUMO machinery, viral DNA replication, and Rep ATPase acitvity. In particular, the third position of the motif was important for each of these activities. We thus uncover a novel mechanism on how geminiviruses recruit the SUMO machinery likely to their own need.

Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

The ubiquitin-like modifying peptide SMALL UBIQUITIN-LIKE MODIFIER (SUMO) has become a known modulator of the plant response to multiple environmental stimuli. A common feature of many of these external stresses is the production of reactive oxygen species (ROS). Taking into account that SUMO conjugates rapidly accumulate in response to an external oxidative stimulus, it is likely that ROS and sumoylation converge at the molecular and regulatory levels. In this study, we explored the SUMO-ROS relationship, using as a model the Arabidopsis (Arabidopsis thaliana) null mutant of the major SUMO-conjugation enhancer, the E3 ligase SAP AND MIZ 1 (SIZ1). We showed that SIZ1 is involved in SUMO conjugate increase when primed with both exogenous and endogenous ROS. In siz1, seedlings were sensitive to oxidative stress imposition, and mutants accumulated different ROS throughout development. We demonstrated that the deregulation in hydrogen peroxide and superoxide homeostasis, but not of singlet O-2 (O-1(2)), was partially due to SA accumulation in siz1. Furthermore, transcriptomic analysis highlighted a transcriptional signature that implicated siz1 with O-1(2) homeostasis. Subsequently, we observed that siz1 displayed chloroplast morphological defects and altered energy dissipation activity and established a link between the chlorophyll precursor protochlorophyllide and deregulation of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE A (PORA), which is known to drive overproduction of O-1(2). Ultimately, network analysis uncovered known and additional associations between transcriptional control of PORA and SIZ1-dependent sumoylation. Our study connects sumoylation, and specifically SIZ1, to the control of chloroplast functions and places sumoylation as a molecular mechanism involved in ROS homeostatic and signaling events. Protein modification by sumoylation and reactive oxygen species are reciprocally regulated in Arabidopsis, with the sumoylation SIZ1 enzyme playing a critical role via control of gene expression.

Being obligate intracellular parasites, viruses rely on the host molecular machinery to replicate and spread. Geminiviruses are a family of plant viruses with circular single-stranded DNA genomes, causal agents of devastating diseases in crops worldwide. None of the geminivirus-encoded proteins is a DNA polymerase, and geminiviral replication, which occurs in the nuclei of infected cells, completely relies on the plant DNA replication machinery. In a first step, the viral ssDNA has to be converted into a double-stranded intermediate, which is then replicated by rolling-circle replication and recombination-dependent replication, producing multiples copies of the original viral genome. Only one viral protein, the replication-associated protein (Rep), is required for the replication of viral DNA: Rep reprograms the cell cycle, recruits the host DNA replication machinery to the viral genome, and mediates the nicking and rejoining events required for the initiation of replication and release of newly synthesized molecules. Another viral protein, C3, plays an ancillary role in viral DNA replication, acting as an enhancer in this process through a mechanism that is not fully understood. In this chapter, we will summarize our current understanding of the geminivirus multiplication in the nuclei of infected plants, including cell cycle reprogramming and viral DNA replication, and explore the open questions in this field.

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Geminiviruses are causal agents of devastating diseases in crops. Geminiviruses have circular single-stranded (ss) DNA genomes that are replicated in the nucleus of the infected plant cell through double-stranded (ds) DNA intermediates by the plant DNA replication machinery. Which host DNA polymerase mediates geminiviral multiplication, however, has so far remained elusive. Here, we show that subunits of the nuclear replicative DNA polymerases α and δ physically interact with the geminivirus-encoded replication enhancer protein, C3, and that these polymerases are required for viral replication. Our results suggest that, while DNA polymerase α is essential to generate the viral dsDNA intermediate, DNA polymerase δ mediates the synthesis of new copies of the geminiviral ssDNA genome, and that the virus-encoded C3 may act selectively, recruiting DNA polymerase δ over ε to favour productive replication.

Plants encode numerous intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived effectors or their activity to activate defenses. miRNAs regulate NLR genes in many species, often triggering the production of phased siRNAs (phasiRNAs). Most such examples involve genes encoding NLRs carrying coiled-coil domains, although a few include genes encoding NLRs carrying a Toll/interleukin-1 domain (TNL). Here, we characterize the role of miR825-5p in Arabidopsis, using a combination of bioinformatics, transgenic plants with altered miRNA levels and/or reporters, small RNAs, and virulence assays. We demonstrate that miR825-5p down-regulates the TNL MIST1 by targeting for endonucleolytic cleavage the sequence coding for TIR2, a highly conserved amino acid motif, linked to a catalytic residue essential for immune function. miR825-5p acts as a negative regulator of basal resistance against Pseudomonas syringae. miR825-5p triggers the production from MIST1 of a large number of phasiRNAs that can mediate cleavage of both MIST1 and additional TNL gene transcripts, potentially acting as a regulatory hub. miR825-5p is expressed in unchallenged leaves and transcriptionally down-regulated in response to pathogen-associated molecular patterns (PAMPs). Our results show that miR825-5p, which is required for full expression of PAMP-triggered immunity, establishes a link between PAMP perception and expression of uncharacterized TNL genes.

ER bodies are endoplasmic reticulum-derived organelles present in plants belonging to the Brassicales order. In Arabidopsis thaliana, ER bodies are ubiquitous in cotyledons and roots and are present only in certain cell types in rosette leaves. However, both wounding and jasmonic acid treatment induce the formation of ER bodies in leaves. Formation of this structure is dependent on the transcription factor NAI1. The main components of the ER bodies are β-glucosidases (BGLUs), enzymes that hydrolyze specialized compounds. In Arabidopsis, PYK10 (BGLU23) and BGLU18 are the most abundant ER body proteins. In this work, we found that ER bodies are downregulated as a consequence of the immune responses induced by bacterial flagellin perception. Arabidopsis mutants defective in ER body formation show enhanced responses upon flagellin perception and enhanced resistance to bacterial infections. Furthermore, the bacterial toxin coronatine induces the formation of de novo ER bodies in leaves and its virulence function is partially dependent on this structure. Finally, we show that performance of the polyphagous beet armyworm herbivore Spodoptera exigua increases in plants lacking ER bodies. Altogether, we provide new evidence for the role of the ER bodies in plant immune responses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Background Small RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics (MIMs or TMs), Short Tandem Target Mimic (STTMs), or molecular SPONGES (SPs). Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches. Results We have developed a vector and a cloning strategy that significantly reduces the number of steps required for the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREEN-DLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette that is itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccd B cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents. Conclusions Our streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.

Geminiviruses (viruses with circular, single-stranded DNA genomes) are one of the major groups of plant viruses causing severe economic problems for agriculture worldwide. The control of these pathogens has become a priority to maintain the production of important crops, including cotton, maize, cassava, and other vegetables. Obtaining resistant plants is the most powerful strategy and a key factor to stablish an effective integrated pest management for a robust control. In the last few decades, numerous studies have successfully approached that goal using diverse strategies based on plant variability or on the engineered expression of proteins/RNAs. The increasing knowledge of the mechanisms involved in the geminivirus-plant-vector interactions, in combination with the development of gene editing technology and nanoparticles, draw new and promising strategies for a durable control of these emerging pathogens.

Nowadays, Huanglongbing (HLB) disease, associated with Candidatus Liberibacter asiaticus (CLas), seriously affects citriculture worldwide, and no cure is currently available. Transcriptomic analysis of host–pathogen interaction is the first step to understand the molecular landscape of a disease. Previous works have reported the transcriptome profiling in response to HLB in different susceptible citrus species; however, similar studies in tolerant citrus species, including Mexican lime, are limited. In this work, we have obtained an RNA-seq-based differential expression profile of Mexican lime plants challenged against CLas infection, at both asymptomatic and symptomatic stages. Typical HLB-responsive differentially expressed genes (DEGs) are involved in photosynthesis, secondary metabolism, and phytohormone homeostasis. Enrichment of DEGs associated with biotic response showed that genes related to cell wall, secondary metabolism, transcription factors, signaling, and redox reactions could play a role in the tolerance of Mexican lime against CLas infection. Interestingly, despite some concordance observed between transcriptional responses of different tolerant citrus species, a subset of DEGs appeared to be species-specific. Our data highlights the importance of studying the host response during HLB disease using as model tolerant citrus species, in order to design new and opportune diagnostic and management methods.