Lactoferrin-Derived Resistance against Plant Pathogens in Transgenic

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Lactoferrin derived resistance against plant pathogens in transgenic plants Dilip K. Lakshman, Savithiry Natarajan, Sudhamoy Mandal, and Amitava Mitra J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf400756t • Publication Date (Web): 26 Jul 2013 Downloaded from http://pubs.acs.org on August 7, 2013

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Journal of Agricultural and Food Chemistry

Lactoferrin derived resistance against plant pathogens in transgenic plants

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Dilip K. Lakshman1*, Savithiry Natarajan2 , Sudhamoy Mandal3, and Amitava Mitra3*

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Floral & Nursery Plants Research Unit and Sustainable Agricultural Systems Laboratory,

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Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, MD. and 3Department

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of Plant Pathology, University of Nebraska Lincoln, NE, USA.

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ABSTRACT:

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Lactoferrin (LF) is a ubiquitous cationic iron-binding milk glycoprotein which contributes to

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nutrition and exert a broad-spectrum primary defense against bacteria, fungi, protozoa and

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viruses in mammals. These qualities make lactoferrin protein and its antimicrobial motifs highly

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desirable candidates to be incorporated in plants to impart broad-based resistance against plant

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pathogens or to economically produce them in bulk quantities for pharmaceutical and nutritional

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purposes. We have introduced bovine LF (BLF) gene into tobacco (Nicotiana tabacum var

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Xanthi), Arabidopsis (A. thaliana) and wheat (Triticum aestivum) via Agrobacterium-mediated

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plant transformation. Transgenic plants or detached leaves exhibited high levels of resistance

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against the damping off causing fungal pathogens Rhizoctonia solani, and the head blight

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causing fungal pathogen Fusarium graminearum. The LF also imparted resistance to tomato

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plants against a bacterial pathogen, Ralstonia solanacearum. Similarly, other researchers

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demonstrated expression of LF and LF-mediated high quality resistance to several other

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aggressive fungal and bacterial plant pathogens in transgenic plants and against viral pathogens

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by foliar applications of LF or its derivatives. Taken together, these reports demonstrated the

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ACS Paragon Plus Environment


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effectiveness of LF for improving crop quality and its biopharming potentials for

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pharmaceautical and nutritional applications.

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KEYWORDS: Antimicrobial peptide, broad-spectrum disease resistance, biopharming, iron-

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binding glycoprotein, lactoferricin, lactoferrampin, LF1-11, transferrin.

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INTRODUCTION:

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Cultivated soil is rich with a multitude of microflora, some of which are beneficial to soil and

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plant health, others are apparently neutral, and a few are pathogenic to plants. According to

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Sullivan (2004)1 a typical teaspoon of native grassland soil would contain at least 10,000 species

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of microbes of which around 5000 species are fungi. Soilborne plant pathogenic fungi and

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nematodes are the major players of soilborne plant pathogens.2 Considering the diversity of soil

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microflora, it is not surprising that about 90% of the 2000 major diseases of principle crops in the

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United States are caused by soilborne plant pathogens,3 resulting in losses in excess of $4

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billion/year.4 Major soilborne plant pathogenic fungi and fungus-like pathogens (oomycetes) are

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species of Rhizoctonia, Fusarium, Sclerotium, Sclerotinia, Thielaviopsis, Phytophthora,

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Pythium, etc. In contrast, only a few groups of plant pathogenic bacteria are considered to be

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soilborne like Ralstonia solanacearum, causal agent of bacterial wilt of tomato,5 and

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Agrobacterium tumefaciens, the well-studied causal agent of crown gall.6 In addition, some

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filamentous bacteria (i.e., Streptomyces) and a few viruses (i.e. Nepoviruses) are soil

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inhabitants.2

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Unlike aerial plant pathogens, soilborne pathogens are difficult to control without

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aggravating the beneficial rhizospheric microflora, and control using fungicides alone can be

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unreliable at times .7,8 Some pesticides used in commercial vegetable and fruit production to 2


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control soilborne diseases can be highly toxic and deleterious to the environment, and pathogens

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often develop resistance to pesticides.9,10 There are only a very limited fungicides registered for

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horticultural use and no suitable alternatives exist to control pathogens in organic production

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systems. Resistant germplasm against Rhizoctonia solani is unavailable and only very few

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commercial agronomic cultivars are partially resistant to the pathogen. In case of the Fusarium

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head blight (FHB, caused by Fusarium graminearum) of wheat, even the optimal fungicide

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applications may only provide a 50-60% reduction in FHB incidence.11 Use of biological control

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has often proved inconsistent or did not work at all under field conditions.12

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An alternative to natural resistance against plant pathogens is the development of

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genetically engineered resistance by incorporation of disease altering or pathogen suppressing

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genes into plants. Currently transgenic crops are commercially grown in at least 25 countries,13

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and transgenic disease-resistant plants represent approximately 10% of the total number of

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approved field trials in North America.14 There are three important strategies employed for

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transgenic resistance. e.g., (a) direct interference with pathogenicity or inhibition of pathogen

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physiology, (b) manipulation of the natural induced host defense, and (c) pathogen mimicry or

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pathogen-derived resistance (PDR) where the plant is designed to express important,

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recognizable features of the pathogen.13,15

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The majority of transgenic trials against bacteria and fungi involve crops expressing

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antimicrobial proteins, which imparts resistance against a broad range of pathogens.16 One such

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antimicrobial peptide function is derived from lactoferrin (LF), a globular milk protein known to

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nourish and defend newborns against fungal, bacterial and viral pathogens. In addition to its use

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for transgenic resistance, recombinant LF (RLF) has been produced in filamentous fungi,

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economic plants, and animal systems for pharmaceutical and nutritional (nutraceutical) 3



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purposes.17-26 The focus of this communication is to review current status on LF-mediated

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transgenic resistance against plant pathogens. While a major emphasis has been placed on

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researches on plant disease resistance conducted by our group, an attempt has also been made to

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summarize all relevant and current literatures on the subject. Moreover, the readers may check

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recent reviews on milk protein-derived antimicrobial peptides27-29 as well as transgenic

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production of RLF in plants, cell cultures, fungi, and mammals for nutraceutical purposes.30,31

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LACTOFERRIN: A MILK PROTEIN WITH MULTIPLE ANTIMICROBIAL

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PEPTIDES

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Lactoferrin is a protein present in milk, tears, saliva, mucus membrane of most mammals, and it

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plays a major role in the immune system of newborns.30 LF is a cationic iron-binding

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glycoprotein of 80 kDa belonging to the transferrin family. LF may have role in iron absorption

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and/or excretion and in gastric health of newborns.32 It has anti-inflammatory and wound healing

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properties , detoxicant, antioxidant and anticancer activities.33-34 Another prominent property of

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LF is its potent activity against a wide range of microorganisms including both gram-negative

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and gram-positive bacteria, as well as fungi and viruses.35 LF consists of two globular domains,

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namely the N- and C-lobe and each lobe has two sub-domains (i.e., N1, N2, C1, C2,

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respectively). 36 The respective domains create one iron binding site on each lobe. The two lobes

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are connected by an α-helix hinge. Although several peptides with antimicrobial activities have

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been identified in LF, the three most characterized are the LF1-11, lactoferricin and

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lactoferrampin - all of them are present in close proximity to each other in the N-terminal lobe of

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bovine LF (BLF) (Figure 1) or human LF (HLF). The lactoferricin and lactoferrampin can be

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released by proteolytic cleavage in the gastrointestinal tracts of mammals. 37 All the three 4


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peptides have higher pI values, hydrophobic and likely interact with negatively charged cellular

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elements like lipopolysaccharides (LPS), DNA, lysozyme, proteoglycans, etc. Lactoferricin has a

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N-terminal amphipathic helix connected to a ߚ-strand with a loop and held together with a

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disulphide bond. It is exposed to the outer surface of the N-lobe of LF and act by disrupting the

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permeability of bacterial cytoplasmic membrane38 . A comparison of antimicrobial activities of

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lactoferricin motifs derived from human, cow, goat and mouse indicated that bovine lactoferricin

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has the highest antimicrobial activity 39 . Similarly, lactoferrampin has an amphipathic α-helix

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structure with a C-terminal tail. It binds to bacterial membrane and causes its disruption40, 41.

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LACTOFERRIN INDUCES PLANT RESISTANCE AGAINST Rhizoctonia solani

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The basidiomycetous soil borne fungus Rhizoctonia solani, sensu lato (Tele: Thanatephorus

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cucumeris, T. praticola, etc) is known to attack 188 species of higher plants in 32 families,

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including various staple crops, ornamentals and turf grasses. 42.43 Some R. solani isolates infect

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distinct tissues on the same host plant causing multiple diseases. The most economically

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important disease caused by R. solani are pre- and post-emergence damping off, root and crown

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necrosis of seedlings, root rots of carrot and beet, aerial blights on foliage, flower and fruits, head

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blight of cabbage and lettuce, soil rots of vegetables, patch diseases of turfgrasses, and sheath

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blight of rice, etc. 44

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In an agar-gel diffusion assay, transgenically expressed LF from tobacco was found to

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inhibit R. solani 45. In the detached tobacco leaf bioassay, necrotic areas developed around the

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mycelial plug in control leaves which rapidly increased in size each day until the entire leaf

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surface became necrotic 9 days after inoculation. On the contrary, transgenic leaves did not show 5



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any visible necrosis for 5-6 days. A small necrotic area was visible in transgenic leaves on day 7

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and only increased slightly on day nine (Figure 2). However, the transgenic resistance seemed to

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breakdown after 10 days as the necrotic area covered the entire leaf during the next two days

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possibly due to rapid senescence and associated protein degradation in the detached leaves. 45 In

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a damping off bioassay of Arabidopsis seedlings expressing LF, the seeds from two T2

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transgenic Arabidopsis lines were sown in pots containing Rhizoctonia inoculum. Most seedlings

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in the control pots died soon after germination although germination of seeds was comparable for

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both transgenic and control Arabidopsis plants. On the other hand, most transgenic seedlings

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grew normally indicating resistance against Rhizoctonia damping off (Figure 3). Thus, our

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experiment demonstrated near complete protection of Arabidopsis seedlings against pre- and

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post-emergence damping off caused by R. solani within the duration of experiment.

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Rhizoctonia normally attacks the juvenile tissues, bedding plants grown from seeds are

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especially vulnerable to pre-emergence damping off. 46-47 On the other hand, seedlings tend to

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develop resistance or tolerance to the pathogen with age. 48-49 Thus, LF-imparted protection

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against Rhizoctonia during the most vulnerable early stage of seedling developmental seems to

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be significant. It could be envisaged that slowing down the onset and progress of Rhizoctonia by

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transgenic expression of LF along with reduced amount or frequency of pesticide application

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have an advantage to the host plant in terms of epidemiology and management of the disease.

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Since

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LACTOFERRIN INDUCES RESISTANCE AGAINST Fusarium graminearum IN

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TRANSGENIC WHEAT:

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Fusarium head blight (FHB) caused by F. graminearum has emerged as a major threat to wheat

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and barley crops around the world. The disease can occur on all small grain crops when the spore

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of the fungus germinates and infects developing kernels on the wheat head. FHB reduces grain

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yield and quality, and is frequently associated with fungal toxins which are hazardous to the

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health of both humans and animals. 50 The United States Department of Agriculture ranks FHB

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as the worst plant disease to hit the USA since the stem rust (Puccinia graminis) epidemic over

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fifty years ago. 51,52 Improving FHB resistance is a high priority in wheat and barley breeding

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programs. We demonstrated that LF inhibits the growth of F. graminearum both in vitro and in vivo.

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'Bobwhite' was significantly higher in transgenic plants expressing LF compared to the control

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'Bobwhite' and in two non-transformed commercial wheat cultivars, ' Wheaton' and 'ND 2710',

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which are susceptible or tolerant to F. graminearum, respectively (Figure 4). The mean percent

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of infection in LF expressing ‘Bobwhite’ wheat varied from 14 - 46% while the mean percent

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infections in non-transformed 'Bobwhite', 'Wheaton' and 'ND 2710' were 82%, 61% and 39%

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respectively (Table 1). Quantification of the expressed LF protein by ELISA in transgenic wheat

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indicated a positive correlation between the LF gene expression levels and the levels of disease

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resistance. More importantly, DON (deoxynivalenol) levels in five transgenic lines, BLFW- 119,

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-378, -424, -892 and - 1102, were below the 1 ppm limit established by the U.S. Food and Drug

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Administration for finished wheat grain products for human consumption. Under greenhouse

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conditions artificially spray-inoculated control 'Bobwhite' lines had an average of 28.5 ppm

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DON. Although natural infection in Nebraska wheat fields varies widely, we routinely detect

We also demonstrated that the level of resistance in the highly susceptible wheat cultivar

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over 5 ppm DON in mildly infected wheat fields and over 50 ppm in moderately infected wheat

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fields.53

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LACTOFERRIN INDUCES PLANT RESISTANCE AGAINST OTHER FUNGAL

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PATHOGENS

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Thus far, only limited studies have been conducted on LF imparted transgenic resistance against

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other fungal plant pathogens. Takase et al 54 expressed the full-length LF and the N-terminal half

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of the molecule in rice plants under the control of the cauliflower mosaic virus 35S promoter.

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Transgenic plants were failed to show resistance against Pyricularia oryzae (causal agent of rice

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blast fungus). However, Fukuta et al55 recently expressed bovine lactoferricin attached to the

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signal peptide of pathogenesis-related protein (PR-1) in tobacco. The signal peptide is supposed

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to secret lactoferricin to apoplast. The transgenic tobacco demonstrated high resistance against

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Botrytis cinerea (causal agent of fruit rot and other plant disease). In non-transgenic experiments,

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the milk product whey, of which LF is a component, was found to control the powdery mildew

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of grapevine, zucchini and cucumber.56-58

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LACTOFERRIN INDUCES PLANT RESISTANCE AGAINST BACTERIAL

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PATHOGENS

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Mitra and Zhang 19 showed that tobacco (Nicotiana tabacum) calli transformed with human LF

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exhibited much higher antibacterial activity than commercially available purified LF against

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four phytopathogenic species, namely Xanthomonas campestris pv phaseoli, Pseudomonas

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syringae pv phaseolicola, P. syringae pv syringae, and Clavibacter flaccumfaciens pv flaccum 8


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faciens. In addition, transgenic tobacco plants expressing LF demonstrated significant delays of

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bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. 59

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Quantification of the expressed LF protein by enzyme-linked immunosorbent assay in transgenic

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plants indicated a significant positive relationship between LF gene expression levels and the

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levels of disease resistance. Later, the same group 60 demonstrated that transgenic tomato plants

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expressing BLF exhibited early resistance and subsequent susceptibility to R. solanacearum,

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while 44% to 55% of infected plants survived until fruit ripening. The transgene was inherited in

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tomato in a stable Mendelian pattern suggesting a potential new approach for controlling

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bacterial wilt of tomato. 60 Similarly, other researchers have demonstrated that transgenically

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expressed LF imparts increased resistance against Erwinia amylovora (causal agent of fire blight

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) in pear, Burkholderia plantarii (causal agent of rice bacterial seedling blight) in rice,

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Pseudomonas syringae pv. tabaci (causal agent of wildfire disease of tobacco, angular leaf spot)

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in tobacco and P. syringae pv syringae and C. michiganensis in alfalfa. 61,54,24,25

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LACTOFERRIN INDUCED PLANT RESISTANCE AGAINST VIRAL PATHOGENS

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In a report on transgenic experiment with LF to test effectiveness against a plant virus, Takase et

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al 54 have shown that rice seedlings expressing BLF or the N-terminal half of BLF does not

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impart demonstrable resistance against rice dwarf virus. On the contrary, Wang et al 62 has

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recently reported that LF and esterified LF (ELF) impart resistance against tobacco mosaic virus

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(TMV) in tobacco seedlings using half-leaf method. The virus inhibitory effect of ELF was

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higher than LF and both the proteins worked in a dose and time-dependent way. Several

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pathogenesis related protein (PR) genes and defense-related enzymes and chemicals were also

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induced both locally and systemically as a result of LF and ELF applications. Abdelbacki et al 9



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has demonstrated that foliar spray with bovine lactoferricin enhances resistance to tomato leaf

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curl virus (TYLCV) on infected tomato plants. 56

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COMMERCIAL PRODUCTION OF LACTOFERRIN AND ITS DERIVATIVES

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The biopharming potentials and applications of LF and its motifs for nutraceutical uses has been

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reviewed recently 30,31. LF has been expressed in fungi, insects, poultry eggs, various mammalian

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cell culture systems, and transgenic animals, including goats, rabbits, and cows, etc. However, to

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an extent those systems have disadvantages like long development time, expensive purification

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processes, logistics issues and potential zoonotic contaminations. On the other hand, plants are

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often suitable for cost effective production of transgenic proteins. In this context, LF has been

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expressed in rice, potato, tobacco, maize, barley, ginseng, alfalfa, etc. Organ specific expression

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of LF in leaves, fruits, cereal grains, and tubers were obtained by utilizing gene expression with

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organ-specific promoters and protein transport signal sequences. The concerns of cell binding,

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immunogenicity, and nutritional values arising from plant glycosylation patterns, retention of

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terminal amino acid sequences and antimicrobial properties, structure and physio-chemical

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stability, agricultural issues as well as economics of various plant expressed LFs have been

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addressed in many primary reports and discussed in recent reviews.30,31

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LACTOFERRIN IS A MULTIFUNCTIONAL ANTIMICROBIAL PROTEIN

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Agriculturally important crop plants need to be protected from devastating diseases to insure

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food security in a changing world climate. As crop plants are routinely infected by serious 10


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fungal, bacterial and viral pathogens, simultaneous control of multiple pathogen groups is

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invaluable. One recent approach to controlling plant diseases has been to express antimicrobial

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genes in transgenic plants . Lactoferrin (LF) appears to be one of the promising broad-spectrum

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non-plant antimicrobial genes with the potential to control aggressive plant pathogens. LF in

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milk is a component of nutrition and organ development of newborn. Copious amounts of LF can

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be readily detected in milk and other routinely consumed dairy products. 63 It is also released in

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bodily secretions, including saliva, tears, bile and pancreatic fluids, etc. 30 Transgenic expression

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of LF also imparts a broad-based resistance against plant bacterial and fungal pathogens.

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19,45,53,59,60

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viruses. 62, 64 This, together with the fact that LF occurs naturally in the diet of humans, makes it

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a novel candidate to introduce plant resistance against diseases. Thus, LF may be considered

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alone or in combination with other transgenes and practices to manage soil borne plant

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pathogenic fungi and bacteria. However, to exploit the full potentials of LF, it will be important

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to demonstrate that the observed resistance against plant pathogens by LF will hold up under

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field conditions. Also, the risk assessments of LF in biopharming should be adequately

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addressed.65,66 Moreover, the effect of LF-expressing transgenic plants on beneficial

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rhizospheric, phyllospheric and endosymbiotic microflora and the acceptability of LF-transgenic

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cultivars by the end-users need to be evaluated.

Moreover, LF and its motifs and derivatives have been demonstrated to inhibit plant

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AUTHOR INFORMATION

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Corresponding Authors

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*Email: [email protected]. Phone: 1 (301) 504-6413.

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*Email: [email protected]. Phone: 1 (402) 472-7054

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Notes

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The authors declare no competing financial interest.

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ABBREVIATIONS USED

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DON, deoxynivalenol; FHB, Fusarium head blight; LF, lactoferrin; BLF, bovine lactoferrin;

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ELF, esterified lactoferrin; HLF, human lactoferrin; RLF, recombinant lactoferrin; PR,

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pathogenesis related protein; TYLCV, tomato leaf curl virus.

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ACKNOWLEDGEMENTS

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We thank Drs. Margaret Pooler and Kathryn Kamo (Floral & Nursery Plants Research Institute,

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USDA-ARS, Beltsville, MD) for reviewing a draft of the manuscript. Research in the Mitra lab

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is partially supported by grants from the NIFA and Nebraska Dry Bean Commission.

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confers resistance to Ralstonia solanacearum in transgenic tobacco plants. Phytopathology 1998,

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(60) Lee, T. J.; Coyne, D. P.; Clemente, T. E.; Mitra, A. Partial resistance to bacterial wilt in

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transgenic tomato plants expressing antibacterial Lactoferrin gene. J. Am. Soc. Hort. Sci. 2002,

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(61) Malnoy, M.; Venisse, J. S.; Brisset, M. N.; Chevreau, E. Expression of bovine lactoferrin

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cDNA confers resistance to Erwinia amylovora in transgenic pear. Mol Breed. 2003, 12, 231-

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Lactoferrin Levels in Term and Preterm Milk. American College of Nutrition. URL

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(http://www.am-coll-nutr.org/nutrition/lactoferrin-levels-in-milk/) (2010).

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(64) Abdelbacki, A. M.; Taha, S. H; Sitohy, M. Z; Dawood, A. I. A. Hamid M.M;and Rezk, A.A.

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Inhibition of Tomato Yellow Leaf Curl Virus (TYLCV) using whey proteins. Virology

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Journal 2010, 7, 26doi:10.1186/1743-422X-7-26.

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(65) Heinemann, J. A. Human Lactoferrin Biopharming in New Zealand - Scientific Risk

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Assessement. 2008, Constructive Conversations/Kōrero Whakaaetanga Rpt 15.pp. 31.

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(66) Broz, A.; Huang, N.; Unruh, G. Plant-based protein biomanufacturing. Genetic Engineering

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and Biotechnology News. 2013, 33(4)

420

421

Legends:

422

Figure 1. The N-lobe of bovine lactoferrin. The three motifs LF1-11, lactoferrampin, and

423

lactoferricin are shown. The 3-D structure was constructed from the GenBank sequence

424

L19981.1, using the Cn3D software from the National Center for Biotechnology Information .

425 426

Figure 2. Detached leaf assay of the fourth leaf of transgenic tobacco seedlings inoculated with

427

R. solani. A 5 mm mycelial disc was placed in the center of each leaf. The leaves were incubated

428

in a humid chamber under fluorescent light at room temperature. The diameters of necrotic

429

symptom developed around the inoculation discs were recorded on 7 and 9 days after

430

inoculation.

431 432

Figure 3. Seedling damping off Bioassay of twelve days old seedlings of two BLF-Arabidopsis

433

transgenic lines (top and bottom left) and two vector only control Arabidopsis lines (top and 20


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434

bottom right) were inoculated with R. solani inoculum mixed with (0.75% wt/wt) autoclaved

435

potting mix.

436 437

Figure 4. Fungal resistance in transgenic wheat - (A) transgenic and (B,C,D) empty vector

438

control wheat lines. The transgenic line is healthy while control lines show various levels of

439

scab infection following inoculation with F. Graminearum in the greenhouse.

440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 21



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457 458 459

TOC Graphic: The graphic shows that incorporation of a bovine lactoferrin gene in wheat can

460

prevent an economically devastating fungal disease.

461 462 463 464 465 466 467 468 469 470 471 472 473 22


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474 475 476

Figure 1.

477

478 479 480 481 482 483 484 485 486 487 488 489 490 23



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491 492 493

Figure 2.

494 495 496 497 498 499 500 501 502

24


Page 25 of 28


503 504 505

Figure 3.

506 507 508 509 510 511 512 513 25



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514 515 516

Figure 4.

517

518 519 520 521 522 523 524 525 526 527 528 26


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529

Wheat lines

Disease severity (%)

BLFW 119 (L+)

15

BLFW 351 (L+)

46

BLFW 378 (L+)

15

BLFW 424 (L+)

19

BLFW 685 (L+)

32

BLFW 892 (L+)

13

BLFW 1102 (L+)

15

BW (L-)

82

Wheaton (NTC)

61

ND 2710 (NTC)

39

530

L+ = Transgenic expressing lactoferrin ; L- = Vector only transgenic control; NTC = Non-

531

transgenic cultivar

532

Table 1. Disease severities in seven transgenic wheat lines. Transgenic lines and three control

533

wheat varieties were spray-inoculated with a conidial suspension of F. graminearum. Disease

534

severity was calculated as percent of infection in the sprayed heads. Six of the seven transgenic

535

lines showed significant Fusarium head blight resistance compared to two commercial wheat 27



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536

varieties, 'Wheaton', 'ND 2710', and a transgenic 'Bobwhite' control carrying an empty vector.

537

BLFW, transgenic 'Bobwhite' wheat lines; BW, wheat cultivar 'Bobwhite' carrying an empty

538

vector; 'Wheaton' and 'ND2710', susceptible and tolerant wheat breeding lines, respectively. All

539

BLFW lines are significantly different from BW at P < 0.01 by Student’s t-tests (Han et al.

540

2012).

541 542 543 544 545 546 547

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Lactoferrin-Derived Resistance against Plant Pathogens in Transgenic

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