Peña, Leandro

Phloem feeders, such as the psyllid Diaphorina citri ( D. citri ), feed on plants by inserting their stylet bundle followed by probing the apoplast before reaching the phloem. The psyllids secrete watery saliva containing various proteins into the phloem, which can act as effectors to facilitate their feeding or modulate host defense responses. Concomitantly, feeding is the main mode of transmitting the Candidatus Liberibacter asiaticus ( C Las) bacteria to the phloem. C Las produces several effectors that have been hypothesized to contribute to Huanglongbing (HLB) virulence. Here, we aimed to identify putative effector proteins from both D. citri and C Las. To achieve this, we used different omics techniques on different tissues and organs from both plants and insects. More specifically, we performed transcriptomics on the heads of healthy and C Las-infected D. citri and proteomics of artificial diet and of phloem of four different plant species fed on by healthy and C Las-infected D. citri. Subsequently, we used various criteria and bioinformatics tools to predict putative effectors. This resulted in the identification of four proteins from D. citri [ferritin, prisilkin, CG31997-PA, and pterin-4-alpha-carbinolamine dehydratase-like protein (PCBD)] and two Sec-dependent effectors from C Las, CLIBASIA_04560 and CLIBASIA_05320, that were used for further functional studies. The expression of these six proteins in Nicotiana benthamiana modified the ROS burst triggered by flagellin, indicating that they can indeed function as effector proteins in planta . In addition, expression of the psyllid effectors in planta significantly reduced the growth of Pseudomonas syringae pv. tabaci ( Pta ).

Huanglongbing (HLB) is a devastating disease of citrus plants caused by the non-culturable phloem-inhabiting bacterium Candidatus Liberibacter ssp., being Ca. Liberibacter asiaticus (CLas) the most aggressive species. CLas is vectored by the psyllid Diaphorina citri and introduced into sieve cells, establishing a successful infection in all Citrus species. Partial or complete resistance has been documented in the distant relatives Murraya paniculata and Bergera koenigii, respectively, providing excellent systems to investigate the molecular basis of HLB-resistance. It has been shown previously that the first weeks after bacterial release into the phloem are critical for the establishment of the bacterium. In this study, a thorough transcriptomic analysis of young flushes exposed to CLas-positive and negative psyllids has been performed in Citrus × sinensis, as well as in the aforementioned resistant species, along the first eight weeks after exposure. Our results indicate that the resistant species do not deploy a classical immunity response upon CLas recognition. Instead, transcriptome changes are scarce and only a few genes are differentially expressed when flushes exposed to CLas-positive and negative psyllid are compared. Functional analysis suggests that primary metabolism and other basic cellular functions could be rewired in the resistant species to limit infection. Transcriptomes of young flushes of the three species are very different, supporting the existence of distinct biochemical niches for the bacterium. These findings suggest that both intrinsic metabolic inadequacies to CLas survival, as well as inducible reprogramming of physiological functions upon CLas recognition, could orchestrate together restriction of bacterial multiplication in these resistant hosts.

Huanglongbing (HLB), the most destructive citrus disease, is associated with unculturable, phloem-limited Candidatus Liberibacter species, mainly Ca. L. asiaticus (Las). Las is transmitted naturally by the insect Diaphorina citri. In a previous study, we determined that the Oceanian citrus relatives Eremocitrus glauca, Microcitrus warburgiana, Microcitrus papuana, and Microcitrus australis and three hybrids among them and Citrus were full-resistant to Las. After 2 years of evaluations, leaves of those seven genotypes remained Las-free even with their susceptible rootstock being infected. However, Las was detected in their stem bark above the scion-rootstock graft union. Aiming to gain an understanding of the full-resistance phenotype, new experiments were carried out with the challenge-inoculated Oceanian citrus genotypes through which we evaluated: (1) Las acquisition by D. citri fed onto them; (2) Las infection in sweet orange plants grafted with bark or budwood from them; (3) Las infection in sweet orange plants top-grafted onto them; (4) Las infection in new shoots from rooted plants of them; and (5) Las infection in new shoots of them after drastic back-pruning. Overall, results showed that insects that fed on plants from the Oceanian citrus genotypes, their canopies, new flushes, and leaves from rooted cuttings evaluated remained quantitative real-time polymerase chain reaction (qPCR)-negative. Moreover, their budwood pieces were unable to infect sweet orange through grafting. Furthermore, sweet orange control leaves resulted infected when insects fed onto them and graft-receptor susceptible plants. Genomic and morphological analysis of the Oceanian genotypes corroborated that E. glauca and M. warburgiana are pure species while our M. australis accession is an M. australis × M. inodora hybrid and M. papuana is probably a M. papuana × M. warburgiana hybrid. E. glauca × C. sinensis hybrid was found coming from a cross between E. glauca and mandarin or tangor. Eremocitrus × Microcitrus hybrid is a complex admixture of M. australasica, M. australis, and E. glauca while the last hybrid is an M. australasica × M. australis admixture. Confirmation of consistent full resistance in these genotypes with proper validation of their genomic parentages is essential to map properly genomic regions for breeding programs aimed to generate new Citrus-like cultivars yielding immunity to HLB.

The Asian citrus psyllid, Diaphorina citri, is the vector of the bacterium “Candidatus Liberibacter asiaticus” (Las), associated with the devastating, worldwide citrus disease huanglongbing. In order to explore the molecular interactions of this bacterium with D. citri during the vector acquisition process, cDNA libraries were sequenced on an Illumina platform, obtained from the gut of adult psyllids confined in healthy (H) and in Las-infected young shoots (Las) for different periods of times (I = 1/2 days, II = 3/4 days, and III = 5/6 days). In each sampling time, three biological replicates were collected, containing 100 guts each, totaling 18 libraries depleted in ribosomal RNA. Reads were quality-filtered and mapped against the Chinese JXGC Las strain and the Floridian strain UF506 for the analysis of the activity of Las genome and SC1, SC2, and type 3 (P-JXGC-3) prophages of the studied Las strain. Gene activity was considered only if reads of at least two replicates for each acquisition access period mapped against the selected genomes, which resulted in coverages of 44.4, 79.9, and 94.5% of the JXGC predicted coding sequences in Las I, Las II, and Las III, respectively. These genes indicate an active metabolism and increased expression according to the feeding time in the following functional categories: energy production, amino acid metabolism, signal translation, cell wall, and replication and repair of genetic material. Pilins were among the most highly expressed genes regardless of the acquisition time, while only a few genes from cluster I of flagella were not expressed. Furthermore, the prophage region had a greater coverage of reads for SC1 and P-JXGC-3 prophages and low coverage in SC2 and no indication of activity for the lysis cycle. This research presents the first descriptive analysis of Las transcriptome in the initial steps of the D. citri gut colonization, where 95% of Las genes were active.

Huanglongbing is a highly destructive citrus disease associated with “Candidatus Liberibacter asiaticus” (Las), a phloem−limited and non-culturable bacterium, naturally transmitted by the psyllid Diaphorina citri. Although diverse approaches have been used to understand the molecular mechanisms involved in the pathogen–host interaction, such approaches have focused on already infected and/or symptomatic plants, missing early events in the initial days post-inoculation. This study aimed to identify the time course of Las multiplication and whole-plant colonization immediately following inoculation by infected psyllids feeding for 2 days. Thus, the experimental approach was to track Las titers after psyllid inoculation in new shoots (NS) of Citrus × sinensis (susceptible), Murraya paniculata (partially resistant), and Bergera koenigii (fully resistant). Soon after psyllid removal, Las titers dropped until the 10–12th days in all three species. Following this, Las titers increased exponentially only in C. × sinensis and M. paniculata, indicating active bacterial multiplication. In C. × sinensis, Las reached a stationary phase at ∼5 log Las cells/g of tissue from the 40th day onward, while in M. paniculata, Las increased at a lower rate of up to ∼3 log Las cells/g of tissue between the 40th and 60th days, decreasing gradually thereafter and becoming undetectable from the 160th day onward. In B. koenigii, Las titers decreased from the start and remained undetectable. In C. × sinensis, an average of 2.6 log of Las cells/g of tissue was necessary for Las to move out of 50% of the NS in 23.6 days and to colonize the rest of the plant, causing a successful infection. Conversely, the probability of Las moving out of the NS remained below 50% in M. paniculata and zero in B. koenigii. To our knowledge, this is the first study on Las dynamics and whole-plant colonization during the earliest stages of infection. Identification of critical time-points for either successful multiplication or Las resistance may help to elucidate initial events of Las–host interactions that may be missed due to longer sampling intervals and at later stages of infection.

Huanglongbing (HLB) is the most destructive, yet incurable disease of citrus. Finding sources of genetic resistance to HLB-associated ‘CandidatusLiberibacter asiaticus’ (Las) becomes strategic to warrant crop sustainability, but no resistantCitrusgenotypes exist. SomeCitrusrelatives of the family Rutaceae, subfamily Aurantioideae, were described as full-resistant to Las, but they are phylogenetically far, thus incompatible withCitrus. Partial resistance was indicated for certain cross-compatible types. Moreover, other genotypes from subtribe Citrinae, sexually incompatible but graft-compatible withCitrus, may provide new rootstocks able to restrict bacterial titer in the canopy. Use of seedlings from monoembryonic species and inconsistencies in previous reports likely due to Las recalcitrance encouraged us to evaluate more accurately theseCitrusrelatives. We tested for Las resistance a diverse collection of graft-compatible Citrinae species using an aggressive and consistent challenge-inoculation and evaluation procedure. Most Citrinae species examined were either susceptible or partially resistant to Las. However,Eremocitrus glaucaand Papua/New GuineaMicrocitrusspecies as well as their hybrids and those withCitrusarose here for the first time as full-resistant, opening the way for using these underutilized genotypes as Las resistance sources in breeding programs or attempting using them directly as possible new Las-resistantCitrusrootstocks or interstocks.

Murraya paniculata and Swinglea glutinosa are aurantioid hosts of the Asian citrus psyllid (ACP) Diaphorina citri, the principal vector of ‘Candidatus Liberibacter asiaticus’ (Las). Las is the pathogen associated with huanglongbing (HLB), the Asian form of which is the most devastating disease of Citrus species and cultivars (Rutaceae: Aurantioideae). M. paniculata is a common ornamental and S. glutinosa is grown as an ornamental, a citrus rootstock, and a hedgerow fence plant. Because of the uncertain status of these plants as reservoirs of Las, a series of cross-inoculation bioassays were carried out in different environments, using infected Valencia sweet orange (Citrus × aurantium) infected shoot tops as a source of inoculum and D. citri nymphs and adults reared on M. paniculata and S. glutinosa to inoculate pathogen-free Valencia orange plantlets. In contrast to sweet orange, Las was more unevenly distributed and reached much lower titers in M. paniculata and S. glutinosa. Infections in M. paniculata and S. glutinosa were also transient. Very few insects that successfully acquired Las from M. paniculata and S. glutinosa were able to transmit the pathogen to healthy citrus. Transmission rates were low from M. paniculata (1.0%) and S. glutinosa (2.0%) and occurred only in a controlled environment highly favorable to Las and ACP using 10-day-old adults that completed their life cycle on Las-positive plants. Our study showed that in HLB-endemic areas, M. paniculata and S. glutinosa can be deemed as epidemiologically dead-end hosts for Las and are not important alternative hosts of the pathogen for transmission to citrus. However, under a combination of conditions highly favorable to Las infection and transmission and in the absence of effective quarantine procedures, these plants could eventually serve as carriers of Las to regions currently free from HLB.

AbstractProduction of citrus, the main fruit tree crop worldwide, is severely threatened by Huanglongbing (HLB), for which as yet a cure is not available. Spread of this bacterial disease in America and Asia is intimately connected with dispersal and feeding of the insect vector Diaphorina citri, oligophagous on rutaceous host plants. Effective control of this psyllid is an important component in successful HLB management programs. Volatiles released from the non-host guava have been shown to be repellent to the psyllid and to inhibit its response to citrus odour. By analysing VOC emission from guava we identified one volatile compound, (E)-β-caryophyllene, which at certain doses exerts a repellent effect on D. citri. Non-host plant rejection mediated by (E)-β-caryophyllene is demonstrated here by using Arabidopsis over-expression and knock-out lines. For the first time, results indicate that genetically engineered Arabidopsis plants with modified emission of VOCs can alter the behaviour of D. citri. This study shows that transgenic plants with an inherent ability to release (E)-β-caryophyllene can potentially be used in new protection strategies of citrus trees against HLB.