Phenotypic and genotypic screening for rust resistance in common bean germplasm in Uganda

Peer Revie

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.).

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm

Heatmap of expression (FPKM) for nine genes encoding NB-ARC domains that were differentially expressed between resistant and susceptible near isogenic lines at 0, 24, 72 and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.

<p>Expression for these nine genes ranged from 0 to 41 FPKM.</p

Genes encoding transcription factors differentially expressed between resistant and susceptible near isogenic lines at 24, 72 and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.

<p>Genes encoding transcription factors differentially expressed between resistant and susceptible near isogenic lines at 24, 72 and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.</p

Genes encoding NB-ARC and leucine-rich repeat domains that were differentially expressed between resistant and susceptible near isogenic lines at 72 and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.

<p>Genes encoding NB-ARC and leucine-rich repeat domains that were differentially expressed between resistant and susceptible near isogenic lines at 72 and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.</p

Number of genes up-regulated in resistant (R) and susceptible (S) near isogenic lines at 0, 24, 72, and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.

<p>Number of genes up-regulated in resistant (R) and susceptible (S) near isogenic lines at 0, 24, 72, and 96 hours post inoculation with <i>Colletotrichum lindemuthianum</i> race 73.</p

Expression patterns of Phvul.001G241300 that encoding a Hs1pro-1 protein in resistant and susceptible near isogenic lines inoculated with <i>Colletotrichum lindemuthianum</i> race 73.

<p>Expression patterns of Phvul.001G241300 that encoding a Hs1pro-1 protein in resistant and susceptible near isogenic lines inoculated with <i>Colletotrichum lindemuthianum</i> race 73.</p

Transcriptome Profiling of the <i>Phaseolus vulgaris</i> - <i>Colletotrichum lindemuthianum</i> Pathosystem

<div><p>Bean (<i>Phaseolus vulgaris</i>) anthracnose caused by the hemi-biotrophic pathogen <i>Colletotrichum lindemuthianum</i> is a major factor limiting production worldwide. Although sources of resistance have been identified and characterized, the early molecular events in the host-pathogen interface have not been investigated. In the current study, we conducted a comprehensive transcriptome analysis using Illumina sequencing of two near isogenic lines (NILs) differing for the presence of the <i>Co-1</i> gene on chromosome Pv01 during a time course following infection with race 73 of <i>C</i>. <i>lindemuthianum</i>. From this, we identified 3,250 significantly differentially expressed genes (DEGs) within and between the NILs over the time course of infection. During the biotrophic phase the majority of DEGs were up regulated in the susceptible NIL, whereas more DEGs were up-regulated in the resistant NIL during the necrotrophic phase. Various defense related genes, such as those encoding PR proteins, peroxidases, lipoxygenases were up regulated in the resistant NIL. Conversely, genes encoding sugar transporters were up-regulated in the susceptible NIL during the later stages of infection. Additionally, numerous transcription factors (TFs) and candidate genes within the vicinity of the <i>Co-1</i> locus were differentially expressed, suggesting a global reprogramming of gene expression in and around the <i>Co-1</i> locus. Through this analysis, we reduced the previous number of candidate genes reported at the <i>Co-1</i> locus from eight to three. These results suggest the dynamic nature of <i>P</i>. <i>vulgaris–C</i>. <i>lindemuthianum</i> interaction at the transcriptomic level and reflect the role of both pathogen and effector triggered immunity on changes in plant gene expression.</p></div

Transcriptome Profiling of the <i>Phaseolus vulgaris</i> - <i>Colletotrichum lindemuthianum</i> Pathosystem - Fig 1

<p>Disease progression on the susceptible NIL genotype a) 0 hpi b) 24 hpi c) 72 hpi and d) 96 hpi. Water soaked lesions were seen at 72 hpi and plants exhibited necrotic lesions at 96 hpi.</p