Petunia Floral Defensins with Unique Prodomains as Novel Candidates for Development of Fusarium Wilt Resistance in Transgenic Banana Plants

Antimicrobial peptides are a potent group of defense active molecules that have been utilized in developing resistance against a multitude of plant pathogens. Floral defensins constitute a group of cysteine-rich peptides showing potent growth inhibition of pathogenic filamentous fungi especially Fusarium oxysporum in vitro. Full length genes coding for two Petunia floral defensins, PhDef1 and PhDef2 having unique C- terminal 31 and 27 amino acid long predicted prodomains, were overexpressed in transgenic banana plants using embryogenic cells as explants for Agrobacterium–mediated genetic transformation. High level constitutive expression of these defensins in elite banana cv. Rasthali led to significant resistance against infection of Fusarium oxysporum f. sp. cubense as shown by in vitro and ex vivo bioassay studies. Transgenic banana lines expressing either of the two defensins were clearly less chlorotic and had significantly less infestation and discoloration in the vital corm region of the plant as compared to untransformed controls. Transgenic banana plants expressing high level of full-length PhDef1 and PhDef2 were phenotypically normal and no stunting was observed. In conclusion, our results suggest that high-level constitutive expression of floral defensins having distinctive prodomains is an efficient strategy for development of fungal resistance in economically important fruit crops like banana

Ex vivo bioassay for Foc resistance in transgenic banana plants.

<p>p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transgenic plants and untransformed control banana plants hardened in a greenhouse for 2 months were challenged with Foc mass culture. External and internal symptoms of Foc infection on p<i>PhDef1</i>-1301 (A and B) and p<i>PhDef2</i>-1301 (C and D) transgenic and untransformed control banana plants (UC). Untransformed control plants showed yellowing of leaves and wilting whereas transgenic plants showed negligible infection. Corm sections of control plants showed intense discoloration whereas the transgenic plants showed significantly less discoloration of the corm tissue.</p

In vitro bioassay for Foc resistance in transgenic banana plants.

<p>In vitro bioassay was performed wherein p<i>PhDef1</i>-1301 (A and B) and p<i>PhDef2</i>-1301 (C and D) transgenic plants and untransformed control banana plants (UC) were challenged using Foc spore suspension. Four weeks post inoculation, untransformed control plants died whereas transgenic plants survived and showed normal growth.</p

Expression analyses of the transgenic banana plants.

<p>(A) and (B) RT-PCR of selected p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301transgenic lines performed using primers specific to <i>PhDef1</i> and <i>PhDef2</i>. (C) and (D) Northern blot analysis of selected p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transgenic lines performed using FA-MOPS gel. Total RNA isolated from the transgenic leaves was filter hybridized with DIG labeled DNA probes targeted against <i>PhDef1</i> (C) and <i>PhDef2</i> (D) gene. Note the high-level expression of <i>Petunia</i> defensins in the different transgenic lines. rRNA bands stained using ethidium bromide indicate equal loading of total RNA in different lanes.</p

Determination of Copy number and genomic integrity of T-DNA in transgenic banana plants.

<p>(A) Southern analysis to determine the transgene copy number in transgenic banana plants expressing <i>PhDef1</i> (D1₆, D1₁₂, D1₁₆, D1₂₀) and <i>PhDef2</i> (D2₃, D2₄, D2₉, D2₁₁). UC denotes an untransformed control plant. Genomic DNA digested with <i>Kpn</i>I was allowed to hybridize with DIG-labeled probes targeted against hygromycin phosphotransferase gene present in the T-DNA of the two binary vectors used for genetic transformation. Genomic DNA digested with <i>Eco</i>RI and <i>Hin</i>dIII + <i>Kpn</i>I and probed with p<i>PhDef1</i> (B) and p<i>PhDef2</i> (C) coding sequences.</p

Genetic transformation of banana cv. <i>Rasthali</i> with full-length <b><i>Petunia</i></b><b> defensin constructs.</b>

<p> (A) <i>Petunia</i> flower which served as the source of defensin genes. (B) T- DNA region of p<i>PhDef1</i>-1301/p<i>PhDef2</i>-1301 binary vector wherein <i>PhDef1</i> and <i>PhDef2</i> coding sequences were cloned downstream of <i>Zea mays</i> polyubiquitin promoter and upstream of nos (nopaline synthase) 3′ UTR in MCS of pCAMBIA-1301 vector. (C) and (D) Somatic embryos derived from p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transformed cells on embryo induction medium 6 weeks post cocultivation. (E) and (F) Germination of transgenic somatic embryos derived from p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transformed cells on medium supplemented with BAP 10 weeks post cocultivation. (G) and (H) Multiple shoots derived from p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transformed cells on shoot induction medium 14 weeks post cocultivation. (I) and (J) Individual shoots derived from p<i>PhDef1</i>-1301 and p<i>PhDef2</i>-1301 transformed cells on root induction medium supplemented with NAA. (K) Hardening of rooted transgenic plants in the green house.</p

Identification and pathogenicity testing of the Foc isolate.

<p>Foc oval shaped spores (A) and mycelium (B) as observed under microscope. Bars correspond to 25 µm (C) Hardened plants of banana cv. <i>Rasthali</i> showing initial symptoms of Foc race 1 infection 3 weeks post inoculation (INF). An uninfected control banana plant is shown on the extreme left (UINF). (D) Necrotic lesions developed inside the corm subsequent to Foc race 1 inoculation on banana cv. <i>Rasthali</i> (left). An uninfected control banana plant is shown on the right. Foc infected untransformed control plants showing disease symptoms after 2 weeks (E) and 4 weeks (F). Mycelium is seen covering the roots of the infected plant.</p

Microscopic studies of the Foc infection in control and transgenic banana plants.

<p>Untransformed control banana plants infected with Foc showed intense discoloration of corm vascular tissue (A) as compared to corm of uninfected control plants (B). Transgenic plants expressing <i>PhDef1</i> (C) and <i>PhDef2</i> (D) show negligible discoloration of the tissue. Bars correspond to 250 µm.</p