Date of Award

7-5-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Plant & Microbial Biosciences)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

In nature, plants regularly contend with bacterial, viral, and fungal pathogens, which employ strategies that promote pathogen fitness and subsequently elicit disease in the host. Xanthomonads are a group of bacterial phytopathogens that induce disease in an extensive range of host plants. Mechanisms underlying pathogen disease promotion have been understudied in food staple crops such as cassava. Cassava is a starchy root crop susceptible to cassava bacterial blight (CBB) a disease caused by the pathogen Xanthomonas phaseoli pv. manihotis (Xpm). Xpm and other bacterial pathogens use effector molecules to manipulate host genes that promote disease susceptibility. Xpm has specialized effector molecules called transcription activator-like (TAL) effectors that enhance pathogen virulence and promote CBB by directly or indirectly inducing the expression of host susceptibility (S) genes. Two TAL effectors, TAL20 and TAL14 respectively target host genes MeSWEET10a, a sugar transporter, and two putative cassava pectate lyases (MePLLs). However, the function of these S genes in promoting bacterial blight is not fully characterized. This thesis describes work done to enhance understanding of the roles that MeSWEET10a and the MePLLs play in promoting CBB disease susceptibility. First, I developed image analysis-based methods to quantify CBB disease severity in Xpm-infected cassava. Next, I used dual gRNA CRISPR/Cas9-mediated genome editing to develop several MeSWEEET10a mutant lines and examined disease susceptibility in mutants. I found that MeSWEEET10a mutant lines exhibited reduced disease symptoms and therefore were less susceptible to CBB. Furthermore, editing MeSWEEET10a did not appear to detrimentally impact the development or function of cassava flowers where MeSWEEET10a is natively expressed. In addition, I investigated the role of putative MePLLs in promoting cassava bacterial blight. I compared MePLL protein sequence similarity to validated pectate lyases from the literature and found that both MePLL sequences had the conserved domain pectate lyase C (Pel C). Work identifying the MePLLs as TAL14 targeted S genes was previously completed in the cassava cultivar, 60444. I used RT-PCR and bacterial growth assays to confirm if virulence-related phenotypes were observed in a farmer-preferred cassava cultivar, TME419. I generated CRISPR/Cas9 constructs with gRNAs targeting the MePLLs, generated several MePLL transgenic lines in the TME419 background, and characterized mutant genotypes. Overall, this thesis work advances the fundamental understanding of host-pathogen interactions, results in MeSWEEET10a mutants with reduced CBB susceptibility, and provides valuable knowledge and resources to further examine the role of MePLLs in promoting Xpm virulence.

Language

English (en)

Chair and Committee

Rebecca Bart

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