Generation and characterization of nanobodies against the Lotp protein of candidatus ?liberibacter asiaticus?, agent of Citrus greening

SPERAT W; GOMEZ JM; PAGLIALI F; GONZALEZ C; VOJNOV A; IBAÑEZ LI

Ciudad de Buenos Aires

Jornada; IX Jornadas Jóvenes Investigadores FVet UBA; 2019

Facultad de Ciencias Veterinarias UBA

Candidatus ?Liberibacter? spp., all vectored by psyllid insects, are recognized as the etiological agents of four devastating plant diseases: citrus greening (also known as Huanglongbing, or HLB), Zebra Chip disease, Psyllid Yellows and Yellows Decline, which currently threaten and destroy the citrus, potato, tomato and carrot industries, respectively. Candidatus ?Liberibacter asiaticus? (Clas) is one of the three causal agents of the citrus HLB disease and is transmitted by Diaphorina citri, the Asian citrus psyllid. During insect feeding, the bacteria are introduced into the phloem and colonize sieve tubes and, though the plant eventually develops chlorosis and dies, it may remain asymptomatic for months or years. After the onset of the symptoms (smaller, deformed fruit with uneven coloration, leaves and shoots develop yellow patches and branch dieback), citrus producers discard the plants and need to re-plat the affected area. Research of the pathogens is extremely complicated, for only one of the genus members (Liberibacter crescens) can be grown in vitro.Lemon is the most important fruit crop in Argentina, for it makes up most of fruit export. Even though the disease still has not reached the Argentine lemon producers, the development of both research and diagnostic tools are of the utmost importance to stay ahead of the disease. In this context, our objective is to develop immunological tools that will allow the study and early diagnosis of HLB. To this end we have selected LotP, a protein from CLas that is overexpressed in planta with respect to the vector, and has been suggested to help with the evasion of the immune system through its interaction with CLas?s GroEL chaperonin homolog. The His-tagged protein was expressed in Escherichia coli, purified and subsequently immunized in Lama glama. After the immunization protocol, the variable fragments of the Lama heavy-chain antibodies (VHHs) were cloned in the pHEN4 phagemid vector to generate an immune library in E. coli; the virus particles expressing the VHH-PIII fusion protein were purified from the supernatant. Selection and characterization of positive binders was done as follows: purified LotP protein was biotinylated in vitro, and added to ELISA plates, which had been previously coated with NeutrAvidin; the phages were pre-incubated with NeutrAvidin to eliminate non binders, and incubated with the target protein; 20, 25 and 30 washes were done with PBST in the first, second and third round of panning, respectively. Lastly, bound phages were eluted, first by incubating with trypsin, and secondly by adding E. coli cells. Serial dilutions of the elutions were plated to observe the enrichment with respect to the negative control. The eluted phages were used to infect E. coli cells and grown overnight to amplify the specific binders. As a next step, ELISA plates were coated with LotP and incubated with purified monoclonal phages to check for positive binders; an unrelated His-tagged protein was used as a negative control. Those clones that had higher affinity with respect to the control were grown and amplified by PCR, followed by a restriction step with the HinfI enzyme. The restriction patterns showed different families of VHH sequences, some clones were selected and sent for sequencing in order to continue characterizing them. In conclusion, we have obtained several possible candidates to test against infected plants and psyllids. These antibodies will be very useful tools to study the bacteria in planta, and could present us with the opportunity to detect the disease before the symptoms manifest.