Figure 1. The systemic acquired resistance (SAR) process from a transgenic rootstock to a non-transgenic scion following infection. Boosting citrus tree immunity to combat HLB

By Manjul Dutt, Juliana Soares and Jude Grosser

and plants such as citrus are generally anchored to a specific location by only when there is another pathogen their roots. Owing to their immobile nature and constant exposure to attack, resulting in a delay in the entire pathogenic microbes, plants are very vulnerable. However, you might be protection process. Lsurprised by how much plants can protect themselves and survive against To facilitate the achievement of multiple adversities encountered during their lifetime. A reason for the survival quicker responses, researchers are ability of plants is their sophisticated and unique defense mechanisms, known as investigating how specifically a plant’s innate immunity, that were developed through evolution. warning signals regulate resistance, the routes that are activated and the SYSTEMIC ACQUIRED plant level like vaccine-mediated products being produced to induce RESISTANCE immunization in humans. defense responses. This information Systemic acquired resistance Although SAR protection does not can be used to engineer plants to (SAR) is a part of the innate immu- last as long as human immunization, directly produce SAR-related products, nity of plants, and it is activated it can last for a week, a month or even thereby keeping the alert system always upon pathogen attack, leading to the an entire season depending on the activated and avoiding any delay in generation of warning signals that plant species. An advantage of SAR resistance responses. translocate throughout the vascular over human vaccines is that it protects The citrus genetic improvement system to notify the entire plant of a plants against a broad spectrum of team at the University of Florida Insti- potential danger. These signals acti- pathogens. However, a disadvantage is tute of Food and Agricultural Sciences vate the plant’s defense responses, that once the effect of SAR disappears, (UF/IFAS) Citrus Research and Educa- providing resistance at the whole the defense responses are reactivated tion Center (CREC) in Lake Alfred is

14 Citrus Industry October 2020 investigating methods to more quickly activate and increase the SAR process in citrus plants. The objective is to confer long-lasting protection, thus Researchers are investigating how specifically making plants tolerant to huanglong- bing (HLB) disease. a plant’s warning signals regulate resistance, the routes that are activated and the products CONVENTIONAL BREEDING Every year, several hundred hybrids being produced to induce defense responses. between selected breeding parents with good potential toward HLB toler- ance are produced in the CREC citrus genetic improvement program. These breeding parents may be derived from possible that enhanced SAR activity The citrus improvement team protoplast fusion (somatic hybridiza- could play a significant role in the developed genetically engineered tion) or prior conventional crosses. development of conventionally bred sweet orange expressing Arabidopsis Crosses are also performed HLB-tolerant rootstocks and scions in NPR1. Trees planted in high HLB between the inedible HLB-tolerant the future. pressure fields showed tolerance to citrus relatives, such as Citrus latipes Candidatus Liberibacter asiaticus. (Khasi papeda) or the edible Citrus GENETIC ENGINEERING Many of these trees have survived australasica (Australian finger lime) The NPR1 protein, a key regulator in a healthy state for many con- with conventional citrus rootstocks of SAR, was discovered in Arabidop- secutive years. Each tree that had and scions. In a recent study, several sis, a small flowering plant related enhanced tolerance to HLB also had putative HLB-tolerant hybrids that to cabbage, cauliflower, broccoli and enhanced expression of the same resulted from these crosses were ana- mustard. This gene has shown great genetic markers that were observed in lyzed. Researchers observed enhanced potential in inducing resistance against the conventionally bred population expression of some genetic markers, pathogens in several crops, including described above. indicating enhanced SAR activity citrus, rice, wheat, soybean, grape, car- These genetically engineered trees within the plant. Thus, it is indeed rot, tomato and strawberry. are still being monitored every year to evaluate the durability of the tolerance effect mediated by the Arabidopsis NPR1 gene. Based on current results, this gene seems to be a good candidate for engineering durable resistance carden to HLB. The citrus improvement We keep you growing... team has also identified other SAR- inducing genes from other crop plants that enhance tolerance to HLB. Engi- neered plants with these gene(s) are also being evaluated in a high disease pressure field site and have shown promising results. In citrus, there is already evidence that plant signals can move from rootstock to scion. This was achieved when a transgenic rootstock expressing the FT early flowering gene induced earlier flowering in grafted juvenile non-transgenic scions. SAR signals are known for their ability to promptly move throughout the plant. There- fore, the citrus improvement team hypothesizes that these molecules are also going to easily move from the engineered rootstocks, eliciting a rapid SPECIALIZED RISK MANAGEMENT FOR THE AGRICULTURE INDUSTRY response to HLB development in the • Carden & Associates, Inc. scion and allowing graft transmissible 888.296.7533 movement of SAR-enhancing signaling www.cardeninsurance.com molecules (Figure 1, page 14). In this scenario, an engineered

16 Citrus Industry October 2020 scion would not be required. The citrus NPR1 gene to become func- to include a value-added trait. This fruit or juice from the tree might not tional, negating the need for the technique could be used to potentially be considered a genetically modified introduction of a foreign DNA. To transfer one or more tolerance gene(s) organism (GMO) at all if GMO pro- resolve this issue, a powerful tech- from an HLB-tolerant citrus relative, teins are not detectable. Time-tested nique called CRISPR-Cas9, which can preserving cultivar integrity without rootstocks (genetically modified) and target specific negative regulators and adding the negative traits that are scions can be used in this approach, silence them, is being utilized. More commonly observed following conven- and this would have a positive impact details on the CRISPR-Cas9 process tional breeding. on consumer acceptance. The cit- will be discussed in a future issue of The genetic modification of citrus rus improvement team is currently this magazine. rootstocks using SAR-related genes is exploring this mechanism to provide also an interesting approach that can durable tolerance to regular sweet CONVENTIONALLY be included in citrus improvement orange scions. However, studies of BRED VS. GENETICALLY programs. The defense signals gener- this nature require time because field MODIFIED CITRUS ated using this approach are mostly testing is a slow process, and it will be Both conventional breeding as well mobile and can potentially translocate a few years before conclusive evidence as genetic engineering strategies have from a genetically modified rootstock is obtained. risks and benefits. Conventionally to a non-transgenic scion. Therefore, Interestingly, citrus also has its developed citrus has been around for as explained above, this process can own NPR1 gene. The question thus a long time, and all cultivated citrus be used in engineering rootstocks that arises: Why does a foreign DNA need plants originate from either sexual can potentially induce resistance in to be inserted into citrus when this crosses or mutations. non-transgenic scions. This can result gene is already present in citrus? The Cisgenesis is another technique in the generation of citrus fruits free of simple answer is that there are several gaining traction in recent years. any transgene. genes that actively interfere with the Cisgenesis is a genetic modification expression of citrus NPR1. These system in which a natural gene from Manjul Dutt (manjul@ ufl.edu) is a genes are known as negative regula- a crossable/sexually compatible cul- research assistant scientist, Juliana tors or genetic suppressors. Turning tivar (that could also be transferred Soares is a postdoctoral associate, and off these negative regulators could using traditional breeding techniques) Jude Grosser is a professor — all at the potentially allow the endogenous is inserted into the recipient plant UF/IFAS CREC in Lake Alfred.

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