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Revista mexicana de fitopatología
versión On-line ISSN 2007-8080versión impresa ISSN 0185-3309
Rev. mex. fitopatol vol.39 no.1 Texcoco ene. 2021 Epub 07-Mayo-2021
https://doi.org/10.18781/r.mex.fit.2009-1
Phytopathological notes
Phylogeny of ATP/ADP translocase gene from Candidatus Liberibacter spp., causal agents of HLB
1 Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Campo Experimental Ixtacuaco. Km 4.5 carretera Federal Martínez de la Torre-Tlapacoyan. Tlapacoyan, Veracruz, México. CP. 93650.
The bacteria that cause citrus huanglongbing (HLB), Candidatus Liberibacter spp., are obligate bacteria to the citrus phloem and to different systems of the vector insect, Diaphorina citri, therefore, the genomic approach has been useful to study its pathogenicity mechanisms. This approach has allowed the identification of a homologous copy of the gene coding for the enzyme ATP / ADP translocase, which has the ability to import ATP and nucleotides from the host, causing considerable energy parasitism. This enzyme has been related to the endoparasitic activity of animal and human pathogens more than to phytopathogens. The present work analyzed the evolutionary relationship between the amino acid sequence of ATP / ADP translocase between different species of Ca. Liberibacter and groups such as Ricketssia sp. and Chlamydia sp. Phylogenetic analyzes show that the variation in the sequence of the gene coding for the enzyme is delimited in clades corresponding to the species of Ca. Liberibacter, suggesting that the variation in the enzyme responds to a co-evolutionary process. Also, the phylogeny shows that the closest common ancestor to Ca. Liberibacter could be a non-pathogenic endosymbiont of the genus Ca. Midichloria. Amino acid sequence conservation analysis shows that there are several positions in the sequence that could be related to species variation. The present work offers the hypothesis that the evolutionary origin of the energy parasitism capacity of the causal agents of HLB is a non-pathogenic endosymbiont.
Key words: coevolution; energy parasitism; citrus greening
Las bacterias causantes del huanglongbing (HLB), Canditatus Liberibacter spp., son bacterias obligadas al floema de los cítricos y a diferentes sistemas del insecto vector, Diaphorina citri, por lo tanto, el enfoque genómico ha sido útil para estudiar sus mecanismos de patogenicidad. Dicho enfoque ha permitido identificar una copia homóloga del gen codificante de la enzima ATP/ADP translocasa, la cual tiene la capacidad de importar ATP y nucleótidos desde el hospedante, causando un parasitismo de energía considerable. Esta enzima se ha relacionado con la actividad endoparasítica de patógenos animales y humanos más que con fitopatógenos. El presente trabajo analizó la relación evolutiva entre la secuencia de aminoácidos de la ATP/ADP translocasa entre diferentes especies de Ca. Liberibacter y grupos como Ricketssia sp. y Chlamydia sp. Análisis filogenéticos muestran que la variación en la secuencia del gen codificante de la enzima está delimitada en clados correspondientes a las especies de Ca. Liberibacter, sugiriendo que la variación en la enzima responde a un proceso coevolutivo con los hospederos. Asimismo, la filogenia muestra que el ancestro común más cercano a Ca. Liberibacter podría ser un endosimbionte no patogénico del género Ca. Midichloria. Análisis de conservación de la secuencia de aminoácidos muestran que existen varias posiciones en la secuencia que podrían estar relacionadas con la variación específica de esta enzima dentro de Ca. Liberibacter. Este trabajo presenta la hipótesis de que el origen evolutivo de la capacidad de parasitismo energético del género Ca. Liberibacter, causantes del HLB, es un endosimbionte no patogénico.
Palabras clave: Coevolución; parasitismo de energía; Dragón Amarillo
Huanglongbing (HLB) is currently considered the most important disease that affects citrus production worldwide, since it causes trees to die a few years after infection, and because, up to now, there are no effective and economically viable methods for controlling it nor resistant varieties accessible to citrus producers. The disease is associated with infection produced by bacteria of the genus Candidatus Liberibacter (Ca. L.), so, it has been observed that HLB is caused by Ca. L. africanus in South Africa (Roberts and Pietersen, 2017), by Ca. L. americanus in Brazil (Wulff et al., 2014), and more extensively by Ca. L. asiaticus, for example, in the United States of America (Manjunath et al., 2008) and Mexico (Flores-Sánchez et al., 2015). These bacteria are obligate pathogens in citrus phloem and endosymbionts in different systems of their vector, Diaphorina citri (Pelz-Stelinski and Killiny, 2016; Killiny et al., 2018), and for this reason obtaining axenic crops is very complex and hinders pathological studies (Ha et al., 2019; Merfa et al., 2019). In order to understand the molecular mechanisms that favor the development of HLB, the genome of several Ca. Liberibacter spp. species has been sequenced (Duan et al., 2009; Lin et al., 2013; Fagen et al., 2014; Zheng et al., 2014, 2015; Cai et al., 2018). This has led to the discovery of genes with high pathogenic potential in the interaction with plants. For example, Vahling et al. (2010) detected the presence of an encoding gene for an ATP/ADP functional translocase enzyme and with great affinity to ATP and ADP, which is important because it has been documented that one of the plant responses to HLB infection is the excessive accumulation of ATP (Pitino et al., 2017). This enzyme has been widely related to obligate intracellular endosymbionts, for it has the capacity of obtaining ATP from the host’s cell and import it into its own cell. This mechanism is known as energetic parasitism (Schmitz-Esser et al., 2004).This is especially interesting because this type of parasitic abilities was not previously known in plant pathogens but only in human and animal pathogens (Trentmann et al., 2007). The evolutionary history of this pathogenic trait suggests that the gene originates from an endosymbiont ancestor of the current Ricketssiales group which, tentatively, moves horizontally among other groups of obligate pathogens (Emelyanov, 2007).
The objective of this study was to analyze the evolutionary relationships among ATP/ADP translocase genes of different human and animal pathogens and endosymbionts, as well as those found in the genus Ca. Liberibacter, including the pathogens that cause HLB in citrus. For this, using the amino acids sequence reported by Vahling et al. (2010) as a base, a BLASTp analysis was conducted to identify the protein sequences with the greatest similarity, according to an e-value <0.001 and 90% identity in the Genbank database, belonging to Ca. Liberibacter and groups such as Ricketssia spp. and Chlamydia spp.
Once the amino acid sequences were obtained, a multiple alignment was done using the ClustalW algorithm (gap open=15; gap extend=3) and the Bioedit software (Hall, 1999). Then, the optimal phylogenetic tree was determined by applying the Maximum Parsimony criterion and T.N.T. software (Goloboff et al., 2008); for this, a search was conducted using compound algorithms (ratchet + sectorial search + drift), and a strict consensus tree was built. A bootstrap resampling (1000 iterations) was performed using the software options. Then, a conservation analysis among the amino acid sequences of the studied enzyme was done. In this case, the alignment was analyzed in order to determine conservation in specific sites using Jalview software (Waterhouse et al., 2009); a positional entropy analysis was also conducted using the function:
Where f(b;l) is the frequency with which each amino acid appears in each column: the frequency is minimum when the position is totally conserved and maximum when all the amino acids are equally represented. The analysis was conducted using Bioedit software.
A total of 39 sequences of the ATP/ADP translocase enzyme were used to root the phylogenetic tree, and the sequence of the ATP/ADP translocase enzyme of Arabidopsis thaliana plastids (Genbank CAA89201) was added as an external group. The phylogenetic analysis made it possible to recover eight equally parsimonious trees (L=2502) with which a strict consensus tree was built (Figure 1). The results of the analysis show that all the Ca. Liberibacter spp. sequences are clustered in a single clade (highlighted in blue); this clade matches with the enzyme coming from the Candidatus Midichloria mitochondrii endosymbiont, which is associated with ticks responsible for causing different diseases worldwide and has an interesting characteristic because it is an intramitochondrial pathogenic bacterium (Sassera et al., 2006).
It is worth noting that within the previously mentioned clade there are well defined subclades with a high bootstrap support according to each species of the genus. The hypothetical basal ancestor of the clade is shared between Candidatus Midichloria mitochondrii and Ca. Liberibacter aeuropaeus, which is interesting because both organisms are considered to be non-pathogenic endosymbionts in ticks (Najm et al., 2012) and pear trees (Raddadi et al., 2011; Camerota et al., 2012), respectively. Ca. Liberibacter americanus and Ca. Liberibacter africanus clades are well defined too; they are pathogenic agents causing HLB in Brazil (do Carmo Teixeira et al., 2005; Teixeira et al., 2008) and Africa (Lin et al., 2006). Likewise, based on the presence of Ca. L. solanacearum and Ca. L. asiaticus as well-defined and supported subclades, it is possible to propose two hypotheses: a) the pathogenic capacity of some Ca. Liberibacter species may have developed in a non-pathogenic endophytic ancestor, given that Ca. L. aeuropaeus is very basal within the Ca. Liberibacter clade, and b) the clades that are well defined by species suggest that their pathogenicity coevolved with the hosts.
The sequences of the Ca. Liberibacter clade show a high level of conservation in some regions, and the reason associated with ATP transportation is intact in all of them (data not shown). However, some regions are highly variable, following a pattern depending on the species to which the sequence of the enzyme belongs, which could produce a change in the structural dynamics and the protein function (Liu and Bahar, 2012). Figure 2A shows the sites with the highest level of conservation and those that have the highest variability according to the alignment frequency. The entropy analysis (Figure 2B) shows that several positions of the ATP/ADP translocase enzyme sequence have an entropic level that suggests high variability in other possible sequences, and for this reason, if new species are found in Ca. Liberibacter with non-reported hosts, the sequence variation could be specific to each host. A thorough study of the function and evolution of this gene will provide a better understanding of the mechanisms whereby damage is caused to plants during the development of HLB and will contribute to developing possible disease control strategies. The results of this study suggest that the capacity of Ca. Liberibacter spp. to infect ATP is a characteristic shared with symbionts and endopathogens. However, the sequence of the enzyme responsible for this parasitism varies according to each species within the genus, which suggests coevolution events of the parasitism with the host.
Figure 1 Phylogenetic analysis of the ATP/ADP translocase enzyme. It shows a consensus tree of eight equally parsimonious trees (L=2502). The Ca. Liberibacter clade is highlighted in blue. The nodes with an asterisk show 100% support using Bootstrap (1000 copies). The name of each tree terminal is made up of the access number to the Genbank followed by the initial of the genus and the species. Abbreviations of the genera: CL or CaLib= Candidatus Liberibacter, R=Ricketssia, O=Orientia.
Figure 2 A) Fragment of the alignment of amino acid sequences of the ATP/ADP translocase enzyme. Amino acidic variability of each Ca. Liberibacter species compared to the sequence of Ca. Midichloria mitocondrii. B) Entropy of the positions of each amino acid in the sequence of the Ca. Liberibacter ATP/ADP translocase enzyme. Axis X= Position of the amino acid, axis Y= Entropy of each position.
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Received: September 01, 2020; Accepted: November 06, 2020
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