Currently, the world citrus industry faces one of its greatest challenges in history, the HLB . In Mexico, this disease is caused by the bacterium Candidatus Liberibacter asiaticus (CLas) , pathogen forced to the phloem of citrus  and different systems of the vector psyllid Diaphorina citri . Due to the above, to date it has been impossible to obtain CLas axenic cultures   which make it impossible to perform virulence, pathogenicity tests as well as Koch’s postulates, to confirm the role of the bacteria in the etiology of the disease .
Given the importance of knowing the mechanisms by which CLas causes the disease, the complete genome of the pathogen has been obtained  . This has allowed the study and characterization of various genetic elements of CLas through bioinformatics and experimental tools . Great advances have been made in the understanding of the molecular interaction between CLas and the plant, for example, the presence of a functional ATP translocase enzyme was observed in the genome of the pathogen, which suggests that one of the mechanisms used by CLas is the parasitism of energy from the plant . Moreover, it has been observed that some pathogenic effectors as Las5315 induces an extreme accumulation of starch in the plant, increment the intracellular H2O2 content and suppress the expression of antioxidant genes  . Likewise, the presence of structures of secretory systems in the bacteria has also been observed , which indicates that the use of virulence proteins is highly related to the activation of symptoms in the plant .
Virulence proteins or pathogenicity effectors play a transcendental role in the plant pathogen interaction according to the zigzag model , as these are responsible for breaking the resistance acquired by the host, as well as be targets for the activation of plant resistance . In the case of phytopathogenic bacteria, effectors are released through the Type III Secretory System   however, in the case of CLas, the structures that make up this system are absent, while the elements of the general secretion pathway (GSP/Sectranslocon) are complete , numerous potential proteins with the signal peptide required for their secretion have also been detected in the CLas genome , even some of them have been evaluated by transient expression in model plants and the characteristic symptoms of the disease have been observed , however, most of these proteins are still without knowledge of its function in the interaction between the plant and the pathogen.
Predicting the function of unknown proteins is one of the main current objectives of bioinformatics. One of the most commonly used approaches for the prediction of protein function is the Gene Ontology (GO), which consists in the systematization of three ontologies: 1) the biological process to which the protein is related, 2) the cellular component where the protein is located and 3) the molecular function of the protein  . Therefore, the objective of this work was to perform, through bioinformatic tools, an analysis of the gene ontology of CLas proteins that are potential pathogenicity effectors.
2. Materials and Methods
2.1. Potential Pathogenicity Effectors
The genes reported by Pitino et al.  selected from Candidatus Liberibacter asiaticus strain Psy62  based on the following criteria: 1) presence of a signal peptide in its structure, 2) length less than 250 amino acids and 3) no existing a characterization of the function in NCBI. The presence of copies of these genes was determined in the genomes of Gxpsy strain , ishi-1 strain , A4 strain  and FL17 strain  through BLAST on the NCBI platform.
2.2. Determination of Gene Ontology
In order to predict the possible function of the proteins of the aforementioned genes, the gene ontology (GO) approach was used  and the ontologies related to the Biological Process (BP), Cellular Component (CC), and Molecular Function (MF) were determined. To carry out the search, an approach based on the affinity propagation and the architecture of the domains in the amino acid sequences was used, using the PANDA online software (http://dna.cs.miami.edu/PANDA/) , with the options offered by default. The results were consulted with the Quick GO database (https://www.ebi.ac.uk/QuickGO/term/GO:0016020) of the European Bioinformatics Institute (EMBL-EBI).
2.3. Search for Conserved Domains and Protein Structure
The presence of functional domains was determined in the different sequences of the genes under study. For this, the online MOTIF search software (https://www.genome.jp/tools/motif/) was used to search the database of Preserved Domains (CDD) of the NCBI, for which the offered options were used. In the case that more than one domain was identified, those not related to prokaryotes were discarded and special attention was focused on those that could show virulence relationship in other organisms. The possible structure of the proteins with potential pathogenic activity coming from the bacteria was determined, for this the software Phyre2 (Protein Homology/analogY Recognition Engine V 2.0) was used  and the results were visualized in the software EzMol 1.22 (http://www.sbg.bio.ic.ac.uk/~ezmol/). Given the experimental importance of the CLIBASIA_05315 proteins , a BLAST of the detected domain was performed and a multiple alignment was generated with the obtained sequences, using the Muscle algorithm; a dendrogram based on genetic distance UPGMA was also built in the MEGA X software . The alignment was analyzed for conservation by specific sites using the Jalview software .
3. Results and Discussion
The sixteen proteins identified as potential pathogenicity effectors by Pitinio et al.  were analyzed to determine their possible role in the interaction that causes the HLB disease. From the cellular component analysis, it was observed that nine of the sixteen proteins are tentatively located in the membrane. The prediction of some proteins was very uncertain, for example, the case of CLIBASIA_02470, which only gave suggestions to localize in membrane, however, no data of biological process or relevant molecular function was obtained. Some of the proteins were related to biological processes associated with cellular energy (CLIBASIA_05315, CLIBASIA_00460 and CLIBASIA_03695), which is of great interest because it is considered that energy parasitism is essential in the HLB process . The results of gene ontology are summarized in Table 1.
The CLIBASIA_00460 protein is related to the electron transfer capacity (Table 1) and is present in all the genomes of CLas analyzed (Table 2), so it is very interesting that in the domain analysis a conserved domain PAAR is located (proline-alanine-alanine-arginine) which has been observed binds to the VgrG spike system as a conical extension, forming a type of contractable mechanism similar to that present in bacteriophages , this belongs to the secretory system type VI (T6SS) and gives it the ability to release different types of toxic molecules to the plant , with a high degree of efficiency and specificity . The domain structure observed in our results (Figure 1(a)) is very similar to those that have been presented in other investigations of the PAAR domain in proteins associated with T6SS . These results suggest that the protein CLIBASIA_00460 may be associated with the release of effectors during the development of HLB mediated by T6SS, which has proved to be an essential part in the development process of other diseases in other crops , because it confers advantages in the adaptation and colonization of the hosts .
Table 1. Prediction of the function based on Gene Ontology of potential proteins as pathogenicity effectors of Candidatus Liberibacter asiaticus described by Pitino et al. .
Table 2. Presence of the different genes with pathogenicity effector potential in different genomes of Candidatus Liberibacter asiaticus reported in the literature.
aDuan et al., 2009; bLin et al., 2013; cKatoh et al., 2014; dZheng et al., 2014; eZheng et al., 2015.
Figure 1. Speculative models of the three-dimensional structure of four proteins with potential effector activity of Candidatus Liberibacter asiaticus (a) CLIBASIA_00460, (b) CLIBASIA_05115, (c) CLIBASIA_05315 and (d) CLIBASIA_04040.
The protein CLIBASIA_05115 is related to the biological process of pathogenesis, while membrane location and molecular function related to nucleotide binding are predicted, therefore, this protein is of future interest in the study of the interaction between CLas and the plant (Table 1), however, no conserved domains were detected (Table 2). The structural analysis of the protein showed a high degree of similarity (data not shown) with the invasive protein Bartonella baciliformis b , which suggests a potential role in the pathogenicity of the bacteria.
A case of special interest in the pathogenic interaction of HLB is the protein CLIBASIA_05315, which proved to be located very close to the chloroplasts when transient expression was made in Nicotiana bethamiana , it is also reported that it causes some of the main symptoms of HLB such as chlorosis and starch accumulation in the same model . Therefore, the detection of a domain corresponding to a β carbonic anhydrase (β-CA) is of relevant importance in the understanding of CLas action mechanisms (Table 3).
Table 3. Conserved domains present in genes with potential pathogenicity effectors Candidatus Liberibacter asiaticus.
*Information obtained from https://www.genome.jp/tools/motif/.
The β-CA enzymes are part of various processes in cells, including respiration and photosynthesis, mediating the reversible reaction of CO2-HCO3 . However, it has been observed that these enzymes are related to many other physiological processes such as CO2 fixation, lipid and amino acid biosynthesis, establishment of seedlings and response to stress . The understanding of the role of these enzymes in the activation of resistance to diseases is not yet well understood, however, experimental evidence suggests that they actively participate as a salicylic acid receptor , and generate activation of Acquired Systemic Resistance (SAR)  ; therefore, it is considered that the role of β-CA is related to a protection against oxidative stress in the plant .
Recent studies indicate that the accumulation of ATP and H2O2 in plants infected with HLB is due to a significant increase in the biosynthesis activity of oxidizing compounds related to the protection of the plant and a decrease in the detoxifying elements of the same, for which reason, CLas generates an oxidative stress that damages the cells of the plant , damage in which the protein CLIBASIA_05315 seems to be intricately involved . The fact that said protein contains a domain related to β-CA suggests that its role in the activation of the disease is that of pathogenicity effector  since it somehow breaks the activity of β-CA present in the chloroplast, altering its photosynthetic activity, causing the accumulation of oxidizing compounds and inhibiting their role in the activation of SAR.
Previously, the presence of β-CA in different pathogenic bacteria has been detected , especially in human pathogens  . Additionally, the presence of these enzymes has been observed in pests and pathogens of agricultural importance, for which it represents a possible objective in the development of chemical control strategies . However, the analysis of conservation (Figure 2) and genetic differentiation (Figure 3) clearly show that the domain found in the protein CLIBASIA_05315 is different from that found in other bacteria,
Figure 2. Conservation analysis of each position in the multiple alignment of the β-CA domain found in the protein CLIBASIA_05315 (last row). A high variation in the amino acid sequence is clearly observed compared to the bacteria to which it is compared by BLAST.
Figure 3. UPGMA analysis of the β-CA domain from the protein CLIBASIA_05315 and different bacteria related by BLASTp.
therefore, experimental evidence of the presence of the β-CA domain in the protein CLIBASIA_05315 is necessary, because this could help to understand more fully the mechanisms by which pathogenicity develops in plants with HLB, as well as develop possible mechanisms to control the disease.
In conclusion, the analysis of gene ontology (GO) allowed us to observe that of the sixteen proteins proposed by Pitino et al. , some are tentatively associated with cellular energy, membranes and electron transfer. The detected domains suggest that the presence of β-CA in CLIBASIA_05315 is related to its affinity to the chloroplast and the physiological alteration previously demonstrated. In turn, the PAAR domain in the protein CLIBASIA_05115 suggests the active participation of T6SS during the development of HLB.
Our findings allow us to direct the future research to the study of effectors of the Candidatus Liberibacter asiaticus in plants as a pathogenicity marker and as a molecular blank for development of diseases control strategies.
The research was supported by a grant from Fideicomiso-INIFAP of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (SIGI 2-1.6-1210034811-A-M.2-3).
 Flores-Sánchez, J.L., Aguilera, G.M., Loeza-Kuk, E., López-Arroyo J.I., Domínguez-Monge, S., Acevedo-Sánchez, G. and Robles-García, P. (2015) Pérdidas en Producción inducidas por Candidatus Liberibacter asiaticus en Limón Persa, en Yucatán México. Mexican Journal of Phytopathology, 33, 195-210.
 Ding, F., Duan, Y., Paul, C., Brlansky, R.H. and Hartung, J.S. (2015) Localization and Distribution of “Candidatus Liberibacter asiaticus” in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-Ompa Polyclonal Antibody. PLoS ONE, 10, e0123939.
 Ghanim, M., Achor, D., Ghosh, S., Kontsedalov, S., Lebedev, G. and Levy, A. (2017) “Candidatus Liberibacter asiaticus” Accumulates inside Endoplasmic Reticulum Associated Vacuoles in the Gut Cells of Diaphorina citri. Scientific Reports, 7, Article No. 16945.
 Davis, M.J. and Brlansky, R. (2007) Culturing Fastidious Prokaryotes—Points to Consider When Working with Citrus Huanglongbing or Greening. Proceedings of the Florida State Horticultural Society, 120, 136-137.
 Jain, M., Munoz-Bodnar, A. and Gabriel, D.W. (2017) Concomitant Loss of the Glyoxalase System and Glycolysis Makes the Uncultured Pathogen “Candidatus Liberibacter asiaticus” an Energy Scavenger. Applied and Environmental Microbiology, 83, e01670-17.
 Chen, J., Deng, X., Civerolo, E.L., Lee, R.F., Jones, J.B., Zhou, C., Hartung, J.S., Manjunath, K.L. and Brlansky, R.H. (2011) “Candidatus Liberibacter Species”: Without Koch’s Postulates Completed, Can the Bacterium Be Considered as the Causal Agent of Citrus Huanglongbing (Yellow Shoot Disease)? Acta Phytopathologica Sinica, 41, 113-117.
 Duan, Y., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Vahling, C.M., Gabriel, D.W., Williams, K.P., Dickerman, A., Sun, Y. and Gottwald, T. (2009) Complete Genome Sequence of Citrus Huanglongbing Bacterium, “Candidatus Liberibacter asiaticus” Obtained through Metagenomics. Molecular Plant-Microbe Interactions, 22, 1011-1020.
 Zheng, Z., Sun, X., Deng, X. and Chen, J. (2015) Whole-Genome Sequence of “Candidatus Liberibacter asiaticus” from a Huanglongbing-Affected Citrus Tree in Central Florida. Genome Announcements, 3, e00169-15.
 Coyle, J.F., Lorca, G.L. and Gonzalez, C.F. (2018) Understanding the Physiology of Liberibacter asiaticus: An Overview of the Demonstrated Molecular Mechanisms. Journal of Molecular Microbiology and Biotechnology, 3610, 116-127.
 Vahling, C.M., Duan, Y. and Lin, H. (2010) Characterization of an ATP Translocase Identified in the Destructive Plant Pathogen “Candidatus Liberibacter asiaticus”. Journal of Bacteriology, 192, 834-840.
 Hao, G., Boyle, M., Zhou, L. and Duan, Y. (2013) The Intracellular Citrus Huanglongbing Bacterium, “Candidatus Liberibacter asiaticus” Encodes Two Novel Autotransporters. PLoS ONE, 8, e68921.
 Pitino, M., Allen, V. and Duan, Y. (2018) LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as Ca. Liberibacter asiaticus Infection in Nicotiana benthamiana. Frontiers in Plant Science, 9, 113.
 Silva, M.S., Arraes, F.B.M., Campos, M.A., Grossi-de-Sa, M., Fernandez, D., de Souza Candido, E., Cardoso, M.H., Franco, O.L. and Grossi-de-Sa, M.F. (2018) Review: Potential Biotechnological Assets Related to Plant Immunity Modulation Applicable in Engineering Disease-Resistant Crops. Plant Science, 270, 72-84.
 Tang, X., Xiao, Y. and Zhou, J.M. (2006) Regulation of the Type III Secretion System in Phytopathogenic Bacteria. Molecular Plant-Microbe Interactions, 19, 1159-1166.
 Chang, J.H., Desveaux, D. and Creason, A.L. (2014) The ABCs and 123s of Bacterial Secretion Systems in Plant Pathogenesis. Annual Review of Phytopathology, 52, 317-345.
 Prasad, S., Xu, J., Zhang, Y. and Wang, N. (2016) SEC-Translocon Dependent Extracytoplasmic Proteins of Candidatus Liberibacter asiaticus. Frontiers in Microbiology, 7, e84469.
 Pitino, M., Armstrong, C.M., Cano, L.M. and Duan, Y. (2016) Transient Expression of Candidatus Liberibacter asiaticus Effector Induces Cell Death in Nicotiana benthamiana. Frontiers in Plant Science, 7, 982.
 Huntley, R.P., Sawford, T., Martin, M.J. and O’Donovan, C. (2014) Understanding How and Why the Gene Ontology and Its Annotations Evolve: The GO within UniProt. Gigascience, 3, 4.
 Lin, H., Han, C.S., Liu, B., Lou, B., Bai, J., Deng, C., Civerolo, E.L. and Gupta, G. (2013) Complete Genome Sequence of a Chinese Strain of “Candidatus Liberibacter asiaticus”. Genome Announcements, 1, e00184-13.
 Katoh, H., Miyata, S.I., Inoue, H. and Iwanami, T. (2014) Unique Features of a Japanese “Candidatus Liberibacter asiaticus” Strain Revealed by Whole Genome Sequencing. PLoS ONE, 9, e106109.
 Zheng, Z., Deng, X. and Chen, J. (2014) Whole-Genome Sequence of “Candidatus Liberibacter asiaticus” from Guangdong, China. Genome Announcements, 2, e00273-14.
 Wang, Z., Zhao, C., Wang, Y., Sun, Z. and Wang, N. (2018) PANDA: Protein Function Prediction Using Domain Architecture and Affinity Propagation. Scientific Reports, 8, Article No. 3484.
 Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N. and Sternberg, M.J.E. (2015) The Phyre2 Web Portal for Protein Modeling, Prediction and Analysis. Nature Protocols, 10, 845.
 Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamuraet, K. (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Molecular Biology and Evolution, 35, 1547-1549.
 Waterhouse, A.M., Procter, J.B., Martin, D.M.A., Clamp, M. and Barton, G.J. (2009) Jalview Version 2-A Multiple Sequence Alignment Editor and Analysis Workbench. Bioinformatics, 25, 1189-1191.
 Shneider, M.M., Buth, S.A., Ho, B.T., Basler, M., Mekalanos, J.J. and Leiman, P.G. (2014) PAAR-Repeat Proteins Sharpen and Diversify the Type VI Secretion System Spike. Nature, 500, 350-353.
 Cianfanelli, F.R., Alcoforado, D.J., Guo, M., De Cesare, V., Trost, M. and Coulthurst, S.J. (2016) VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System. PLoS Pathogens, 12, e1005735.
 Bondage, D.D., Lin, J.S., Ma, L.S., Kuo, C.H. and Lai, E.M. (2016) VgrG C Terminus Confers the Type VI Effector Transport Specificity and Is Required for Binding with PAAR and Adaptor-Effector Complex. Proceedings of the National Academy of Sciences, 113, E3931-E3940.
 Kamber, T., Pothier, J.F., Pelludat, C., Rezzonico, F., Duffy, B. and Duffy, T.H.M. (2017) Role of the Type VI Secretion Systems during Disease Interactions of Erwinia amylovora with Its Plant Host. BMC Genomics, 18, 628.
 Coleman, S.A. and Minnick, M.F. (2001) Establishing a Direct Role for the Bartonella bacilliformis Invasion-Associated Locus B (Ia1B) Protein in Human Erythrocyte Parasitism. Infection and Immunity, 69, 4373-4381.
 DiMario, R.J., Clayton, H., Mukherjee, A., Ludwig, M. and Moroney, J.V. (2017) Plant Carbonic Anhydrases: Structures, Locations, Evolution, and Physiological Roles. Molecular Plant, 10, 30-46.
 Medina-Puche, L., Castelló, M.J., Canet, J.V., Lamilla, J., Colombo, L. and Tornero, P. (2017) β-Carbonic Anhydrases Play a Role in Salicylic Acid Perception in Arabidopsis. PLoS ONE, 12, e0181820.
 Klessig, D.F., Choi, H.W. and Dempsey, D.A. (2018) Systemic Acquired Resistance and Salicylic Acid: Past, Present, and Future. Molecular Plant-Microbe Interactions, 31, 871-888.
 Floryszak-Wieczorek, J. and Arasimowicz-Jelonek, M. (2017) The Multifunctional Face of Plant Carbonic Anhydrase. Plant Physiology and Biochemistry, 112, 362-368.
 Pitino, M., Armstrong, C.M. and Duan, Y. (2017) Molecular Mechanisms behind the Accumulation of ATP and H2O2 in Citrus Plants in Response to “Candidatus Liberibacter asiaticus” Infection. Horticulture Research, 4, Article No. 17040.
 Tobal, J.M. and da Silva Ferreira Balieiro, M.E. (2013) Role of Carbonic Anhydrases in Pathogenic Microorganisms: A Focus on Aspergillus fumigatus. Journal of Medical Microbiology, 63, 15-27.
 Stefanucci, A., Angeli, A., Dimmito, M.P., Luisi, G., Del Prete, S., Capasso, C., Donald, W.A., Mollica, A. and Supuran, C.T. (2018) Activation of β- and γ-Carbonic Anhydrases from Pathogenic Bacteria with Tripeptides. Journal of Enzyme Inhibition and Medicinal Chemistry, 33, 945-950.
 Emameh, Z.R., Barker, H., Hytonen, V.P., Tolvanen, M.E.E. and Parkkila, S. (2014) Beta Carbonic Anhydrases: Novel Targets for Pesticides and Anti-Parasitic Agents in Agriculture and Livestock Husbandry. Parasites and Vectors, 7, 403.