Pearl Millet with Enhanced Resistance to Rust and Downy Mildew

ISB News Report November 2006
Pearl Millet with Enhanced Resistance to Rust and Downy Mildew
Janaki Krishna
Pearl millet (Pennisetum glaucum), also commonly known as cumbu, bajra, and cattail millet, is an important grain,
forage, and stover crop, accounting for approximately 50% of the total world production of millet. Pearl millet is grown
on more than 29 million ha. in arid, semi-arid, subtropical, and tropical regions of Asia, Africa, and Latin America, where
it is often a basic staple among the poorest people. Increasingly, pearl millet is grown in non-traditional areas in temperate,
developed countries, where diseases have a larger impact. The major diseases that account for severe crop damage in pearl
millet are downy mildew (Sclerospora graminicola), smut (Moesziomyces penicillariae), ergot (Claviceps fusiformis), and
rust (Puccinia substriata).
Due to substantial annual yield losses, disease resistance has become a high priority for pearl millet breeders. In
addition to conventional methods, genetic engineering techniques are used to enhance disease resistance. Antifungal
proteins and polypeptides have been isolated from diverse groups of organisms, including plants, fungi, bacteria, insects,
and animals (both vertebrates and invertebrates). The mechanisms of action of these proteins are as varied as their sources
and include fungal cell wall polymer degradation, membrane channels and pore formation, damage to cellular ribosomes,
inhibition of DNA synthesis, and inhibition of cell cycle. Genes encoding cell wall degrading enzymes, e.g., chitinases,
antimicrobial peptides, and antifungal proteins, are currently used to create genetically engineered plants with increased
fungal resistance in the field. However, whether these transgenic crops gain acceptance in the marketplace remains to be
seen.
Expression of antifungal proteins in transgenic plants has improved fungal resistance experimentally in wheat and
rice. Researchers from University of Hamburg, Germany, University of Wales, UK, and Australian Centre for Plant
Functional Genomics, Australia, recently demonstrated enhanced fungal resistance in pearl millet to two economically
important diseases, rust and downy mildew, through stable integration of an antifungal gene (afp) from Aspergillus
giganteus. They used two pearl millet genotypes, Manga Nara from the Savannah African Research Institute (SARI),
Ghana, and 7042 from the International Crop Research Institute for Semi-Arid Tropics (ICRISAT), India. The biolistic
transformation of immature zygotic embryos was carried out using two plant expression vectors: vector p35SacS, which
carries the phosphinothricin acetyltransferase (pat) gene from Streptomyces viridochromogenes and confers tolerance to
herbicide ‘BASTA’; and vector pubi2afp, which contains cDNA encoding the antifungal protein (afp).
Immature zygotic embryos of Manga Nara and 7042 were co-bombarded with the afp and pat gene vectors at 1550
psi. Two-week-old plants were selected for further studies on gene integration and expression. Transgenic plants M1, from
the genotype Manga Nara, and 701, from genotype 7042, had transformation rates of 0.15% and 0.13%, respectively.
These plants displayed normal growth, seed set, and fertility. The presence of BASTA and afp genes in T0, T1, and
T2 plants was confirmed by molecular studies, including Southern blot analysis. A 3:1 segregation ratio was found for
BASTA tolerant to sensitive plants in the T1 generation. Integration of afp gene was confirmed by RT-PCR analysis in the
T1 and T2 generations of both M1 and 701 transgenic lines.
In vitro inoculation of afp expressing plants with Puccinia substriata revealed that rust infection of detached leaf
segments from the transformed lines M1 and 701 was significantly reduced when compared to their control wild type
genotypes. When compared with controls, M1 and 701 transgenic lines of pearl millet showed 11.2% and 8.6% disease
severity, respectively, thus demonstrating up to 90% enhancement in disease resistance of transgenic plants.
In all inoculated transgenic plants, pustule formation was delayed in comparison to the control, and the size of P.
substriata pustules on wild type plants was bigger. The in vitro leaf segment inoculation results were further verified
by comparing them with the in vivo inoculation results conducted on whole transgenic homozygous T2 progeny
and wild type progeny. The transgenic plants from M1 and 701 lines showed a high resistance when compared with
corresponding wild types, and only 6 of 52 (13.3%) inoculated M1 plants and 7 of 44 (18.8%) inoculated 701 plants
showed rust pustules formation, whereas 37 of 41 (85.7%) and 21 of 25 (79.7%) were infected among wild type plants
of Manga Nara and 7042, respectively.
Likewise in vivo inoculation of homozygous T2 from transgenic lines M1 and 701 with Sclerospora graminicola
showed an efficient enhancement in downy mildew resistance compared to the corresponding wild types. Out of 166
inoculated MI transgenic plants, only 2 (1.2%) showed disease incidence compared to wild type, whereas 95 of 151
(63%) Manga Nara plants were infected. In the 701 transgenic line, 2 of 237 (0.8 %) inoculated plants showed infection
symptoms compared to 96.9% infected wild type plants (132 of 136).

ISB News Report November 2006
In summary, the researchers report the first demonstration of significant fungal resistance in which the afp gene from
Aspergillus giganteus was stably integrated in two pearl millet genotypes by particle bombardment of immature zygotic
embryos. In vitro and in vivo studies of pearl millet transgenic lines revealed enhanced resistance to the deadly diseases of
rust and downy mildew. The expression of the afp gene in the two pearl millet cultivars Manga Nara and 7042 confirms
the potential of afp for the improvement of disease resistance and offers great promise in plant protection strategies
through genetic engineering. The expression of the afp gene in two cultivars representing different genetic backgrounds
validates its potential for disease resistance strategies.
Source:
Girgi M, Breese WA, Lörz H and Oldach KH. (2006) Rust and downy mildew resistance in pearl millet (Pennisetum glaucum) mediated
by heterologous expression of the afp gene from Aspergillus giganteus. Transgenic Research 15: 313-324
P S Janaki Krishna
Institute of Public Enterprise
Osmania University Campus, Hyderabad, India
jankrisp@yahoo.com