HPE1, an Effector from Zebra Chip Pathogen Interacts with Tomato Proteins and Perturbs Ubiquitinated Protein Accumulation.

The gram-negative bacterial genus Liberibacter includes economically important pathogens, such as 'Candidatus Liberibacter asiaticus' that cause citrus greening disease (or Huanglongbing, HLB) and 'Ca. Liberibacter solanacearum' (Lso) that cause zebra chip disease in potato. Liberibacter pathogens are fastidious bacteria transmitted by psyllids. Pathogen manipulation of the host' and vector's immune system for successful colonization is hypothesized to be achieved by Sec translocon-dependent effectors (SDE). In previous work, we identified hypothetical protein effector 1 (HPE1), an SDE from Lso, that acts as a suppressor of the plant's effector-triggered immunity (ETI)-like response. In this study, using a yeast two-hybrid system, we identify binding interactions between tomato RAD23 proteins and HPE1. We further show that HPE1 interacts with RAD23 in both nuclear and cytoplasmic compartments in planta. Immunoblot assays show that HPE1 is not ubiquitinated in the plant cell, but rather the expression of HPE1 induced the accumulation of other ubiquitinated proteins. A similar accumulation of ubiquitinated proteins is also observed in Lso infected tomato plants. Finally, earlier colonization and symptom development following Lso haplotype B infection are observed in HPE1 overexpressing plants compared to wild-type plants. Overall, our results suggest that HPE1 plays a role in virulence in Lso pathogenesis, possibly by perturbing the ubiquitin-proteasome system via direct interaction with the ubiquitin-like domain of RAD23 proteins.

A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing.

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

Melatonin Is Involved in Citrus Response to the Pathogen Huanglongbing via Modulation of Phytohormonal Biosynthesis.

Huanglongbing (HLB) is a devastating citrus disease worldwide that is putatively caused by Candidatus Liberibacter asiaticus and transmitted by Diaphorina citri Melatonin is a ubiquitously distributed auxin-like metabolite found in both prokaryotes and eukaryotes. In this study, we used integrative metabolomic and transcriptomic approaches to investigate the potential role of melatonin in citrus response against HLB and to understand the relationships between melatonin and the stress-associated phytohormones at molecular and metabolic levels. Melatonin was detected in the leaves of Valencia sweet orange (Citrus sinensis) after derivatization with N-methyl-N-trimethylsilyltrifluoroacetamide using a targeted gas chromatography-mass spectrometry running in selective ion monitoring mode-based method. Ca. L. asiaticus infection and D. citri infestation significantly increased endogenous melatonin levels in Valencia sweet orange leaves and upregulated the expression of its biosynthetic genes (CsTDC, CsT5H, CsSNAT, CsASMT, and CsCOMT). However, infection with Ca. L. asiaticus had a greater effect than did infestation with D. citri Melatonin induction was positively correlated with salicylic acid content, but not that of trans-jasmonic acid. Moreover, melatonin supplementation enhanced the endogenous contents of the stress-associated phytohormones (salicylates, auxins, trans-jasmonic acid, and abscisic acid) and the transcript levels of their biosynthetic genes. Furthermore, melatonin supplementation diminished the Ca. L. asiaticus titer within the infected leaves, which suggests that melatonin might play an antibacterial role against this bacterium and gram-negative bacteria in general. These findings provide a better understanding of the melatonin-mediated defensive response against HLB via modulation of multiple hormonal pathways. Understanding the role of melatonin in citrus defense to HLB may provide a novel therapeutic strategy to mitigate the disease.

Detección de patógenos asociados a la enfermedad punta morada en los cultivos de papa y tomate en Guatemala / Detection of pathogens associated to potato purple top disease in potato and tomato crops in Guatemala

La punta morada es una enfermedad que afecta la producción de algunas especies de solanáceas como la papa y el tomate, causando enrollamiento en las puntas de las hojas con una marcada coloración morada, decaimiento temprano de la planta y en la papa se observa tuberización aérea. Como patógenos asociados a la enfermedad se consideran al fitoplasma BLTVA y la bacteria Candidatus Liberibacter solanacearum. Dada la similitud en la sin-tomatología foliar que generan ambos patógenos, es difícil precisar cuál de ellos está implicado en la enfermedad. En Guatemala, existen reportes de la sintomatología típica de punta morada en las principales zonas productoras de papa y tomate, desconociéndose el agente asociado. La investigación determinó cuál de los dos patógenos reportados está asociados a la enfermedad en 12 municipios productores de papa y/o tomate en el país. Se realizaron ampli-ficaciones de ADN con cebadores específicos para cada patógeno asociado a la enfermedad. Por la alta incidencia del fitoplasma BLTVA en las muestras de papa (73.9%), en comparación a C. Liberibacter solanacearum (26%), este es considerado como el patógeno asociado más importante en papa. En las muestras de tomate, la incidencia del fitoplasma BLTVA (29.8%) y C. Liberibacter solanacearum del (27.6%) fue similar. Además, sobresale el primer reporte de la detección del fitoplasma BLTVA afectando el cultivo de tomate en Guatemala. Se sugiere un monitoreo constante, mediante métodos moleculares, para un diagnóstico certero y establecer medidas de manejo de la enfermedad para evitar su diseminación hacia zonas aún no afectadas.

The potato purple top is a disease that affects the production of some solanaceous species such as potatoes and tomatoes, causing curl at the tips of the leaves with a marked purple coloration, early decay of the plant, and aerial tuberization is observed in the potato. BLTVA phytoplasma and Candidatus Liberibacter solanacearum are considered as pathogens associated with the disease. Given the similarity in foliar symptoms generated by both pathogens, it is difficult to determine which one is involved in the disease. There are reports of the typical potato purple top symptoms in the main potato and tomato producing areas in Guatemala, being unknown the associated agent. The research determined which of the two reported pathogens is associated with the disease in 12 potatoes and/or tomato producing areas in the country. We performed DNA amplification with specific primers for each disease-associated pathogen. Due to the high incidence of BLTVA phytoplasma in potato samples (73.9%), com-pared to C. liberibacter solanacearum (26%), this is considered the most important associated pathogen in potatoes. In tomato samples, the incidence of BLTVA phytoplasma (29.8%) and C. liberibacter solanacearum (27.6%) was similar. Besides, the first report of the detection of the BLTVA phytoplasma affecting tomato cultivation in Gua-temala stands out. Using molecular methods, constant monitoring is suggested for an accurate diagnosis and to establish management measures for the disease to prevent its spread to areas not yet affected.