A promising plant defense peptide against citrus Huanglongbing disease

Citrus fruits and orange juice are beloved components of daily diets, owing to their delicious taste, high levels of vitamin C, folate, and flavanones hesperidin and naringin, and many health benefits (1). Just as we are facing the COVID-19 pandemic, citrus is facing a devastating pandemic of its own, known as Huanglongbing (HLB) or greening disease (2, 3). Citrus production in Florida has decreased by 74% since the first report of HLB in Florida in 2005 (4). There is currently no cure for HLB-positive citrus trees, and those that contract the disease die within a few years (2). Although Candidatus Liberibacter asiaticus (CLas), Ca. L. americanus, and Ca. L. africanus have not been cultured in artificial media, modified Koch’s postulates have been fulfilled to corroborate their status as the causative agents of HLB (2). Liberibacter species belong to the family Rhizobiaceae. The endocytosis capacity of Rhizobiaceae species may have played important roles in the phloem colonization and eventual establishment in psyllid hosts of the ancestor of Liberibacters (3). CLas is the most prevalent HLB pathogen worldwide, and it is mainly transmitted by Asian citrus psyllids (Diaphorina citri). The inability to culture HLB pathogens has significantly hampered our understanding of the virulence mechanisms of HLB and the development of HLB control approaches (3, 5). In PNAS, Huang et al. (6) characterize a heat-stable antimicrobial peptide MaSAMP from HLB-tolerant Microcitrus that both kills CLas and induces plant immunity (Fig. 1), representing a potential breakthrough for HLB control. Because of its dual role, it can be categorized as a host defense peptide (7). This new finding will likely trigger broad interest in identifying and using host defense peptides in other plants.

Fig. 1.

Schematic representation of identification of host defense peptides from HLB resistant/tolerant citrus genotypes and application of host defense peptides to control HLB.

Currently, a strategy comprising psyllid control, inoculum removal, and replantation with HLB-free trees, which interrupts the HLB disease cycle, has been recommended as a practical solution for controlling HLB (2, 3). However, key to the success of such a strategy is large-scale (region-wide) comprehensive implementation (8), which is challenging in execution. Small-scale or noncomprehensive implementation of such a strategy fails to prevent HLB from spreading because of psyllid dispersal and subsequent transmission of CLas (8). Consequently, HLB has become endemic in many citrus-producing regions, such as Florida and southeastern Asia (9). As a phloem-colonizing and systemic pathogen, CLas resembles many chronic human pathogens that infect the respiratory tract, gastrointestinal tract, or blood vessels. Unlike most plant pathogens, CLas that has colonized the phloem is shielded from many environmental stresses, such as ultraviolet (UV) radiation, heat, and drought, that microbes commonly encounter (10), and from chemical treatments. Huang et al. identify an efficient antimicrobial peptide that can reach the phloem to kill CLas, thereby reducing HLB symptoms and preventing new CLas infections, as suggested by the data from greenhouse trials (6).

MaSAMP was identified from HLB-tolerant Australian finger lime (Microcitrus australasica), a close relative of Citrus. A similar antimicrobial peptide was also identified in HLB-tolerant trifoliate orange (Poncirus trifoliata), but it had a lower antimicrobial activity. Most commercial citrus varieties (with a few exceptions, such as Sugar Belle) are susceptible to HLB, owing to their limited genetic diversity (11, 12). However, multiple citrus relatives, such as M. australasica, P. trifoliata, Balsamocitrus dawei, Bergera koenigii, Casimiroa edulis, Clausena excavata, Murraya paniculata, Citrus medica, Eremocitrus glauca, Citrus latipes, Carrizo citrange, Persian lime, and Eureka lemon, have shown tolerance/resistance against HLB. Genetic resistance is the most effective, economic, and, as a result, sustainable approach to crop protection against pests. However, utilization of the HLB-tolerant/resistant genetic materials in traditional breeding has been limited by the long juvenile phase, nucellar embryony, sexual incompatibility, highly heterozygous nature, and male or female sterility of many citrus genotypes (13).

The approach used by Huang et al. (6) to identify antimicrobial peptides from HLB-tolerant/resistant materials probably has only scratched the surface regarding further utilizing these HLB-tolerant/resistant genetic materials for HLB control, such as the discovery of other antimicrobial peptides to synergistically enhance the antimicrobial activity and identifying genes underlying HLB tolerance/resistance (Fig. 1). Multiple nucleotide-binding site and leucine-rich repeat genes that might contribute to the HLB tolerance/resistance of Poncirus have been reported (14). Plant defense regulators and constitutive disease resistance genes have also been suggested to contribute to the HLB tolerance of Poncirus or an HLB-tolerant citrus hybrid (P. trifoliata × Citrus reticulata). Importantly, identification of the gene encoding the host defense peptide (6) and other HLB tolerance/resistance genes opens the door for generating HLB-tolerant/resistant varieties via knock-in modification using CRISPR-Cas technology (Fig. 1) (15). Fortunately, this technology has been successfully used to edit citrus genomes and generate disease-resistant citrus varieties (16–20).

Antimicrobials, such as oxytetracycline and streptomycin, have been approved to control HLB via foliar spray. However, neither antibiotic is able to control HLB when applied via foliar spray, because neither antibiotic reaches an effective concentration in plants to suppress CLas (21, 22). Injection, as a common delivery method for antibiotics to treat bacterial pathogens that infect the respiratory tract, gastrointestinal tract, or blood vessels of humans, is capable of delivering oxytetracycline and streptomycin at effective concentrations in plants (21, 22). However, antimicrobial application via injection has not yet been approved for HLB management. In addition, the antibiotic residue levels in citrus fruit that are approved for human consumption need to be taken into consideration. Conversely, the host defense peptide (6), owing to its citrus origin, may cause less concern in terms of residue levels. It can reach up to 22 μM in the vascular fluid after foliar spray, well above the concentration needed to kill CLas (6). How the host defense peptide (6) becomes efficiently absorbed by citrus plants remains to be determined. Its heat stability seems to contribute to its antimicrobial activity in plants. In contrast, streptomycin is less heat stable, and oxytetracycline is degraded under UV and visible light. Application of oxytetracycline or streptomycin results in subinhibitory antibiotic concentrations that are known to select for antibiotic resistance. Antimicrobial peptides are less prone to resistance development than antibiotics, as their mode of action exploits general but essential structural components of pathogens, such as the cell membrane. In addition to the antimicrobial peptides identified by Huang et al. (6), other approaches to identify antimicrobials against CLas may also provide promising alternatives to control HLB (23).

In PNAS, Huang et al. characterize a heat-stable antimicrobial peptide MaSAMP from HLB-tolerant Microcitrus that both kills CLas and induces plant immunity (Fig. 1), representing a potential breakthrough for HLB control.

In addition to antimicrobial activity, MaSAMP also induces plant defense responses. Plant defense inducers, such as salicylic acid (SA), SA analogs, and brassinosteroids, have been tested to control HLB via inducing plant immunity. However, the induced plant defense is complicated by the SA hydroxylase encoded by CLas, which degrades SA and negatively affects plant defense induction. In addition, CLas encodes other proteins, such as SDE1, SDE15, and 1-Cys peroxiredoxin, to suppress plant immunity. Intriguingly, the plant defense peptide identified by Huang et al. (6) is able to overwhelm the countermeasures of CLas to induce plant immunity. This process is dependent on pathogenesis-related gene 1 (NPR1) and suppressor of the G2 allele of skp1 (SGT1), although the receptor for such recognition remains unknown.

As reported by Huang et al. (6), MaSAMP differs from most known antimicrobial peptides, such as defensins, thionins, lipid transfer proteins, snakins, hevein-like peptides, knottins, and hairpinins, which are cysteine rich and contain <50 amino acids. MaSAMP is not a cystine-rich peptide and contains 67 amino acids, probably representing a new family of antimicrobial peptides. Importantly, its antibacterial activity seems to challenge the view that antimicrobial peptides commonly have low and broad-spectrum activity. Instead, MaSAMP shows superior antibacterial activity compared with streptomycin and specificity against α-proteobacteria (6). Nevertheless, many of its characteristics, such as its minimum bactericidal concentration and minimum inhibitory concentration, and the mechanism underlying its mode of action and specificity, remain to be explored. Mechanistic understanding of its mode of action and specificity might provide cues regarding how to improve activities of antimicrobial peptides. It is also anticipated that the host defense peptide can prevent psyllid acquisition and transmission of CLas, which needs to be tested. The host defense peptide is encoded by Microcitrus that produces edible fruits (6), and can be crossed with Citrus. Thus, it is anticipated that its utilization (either direct application as a synthesized peptide or introduction into elite citrus varieties via CRISPR-Cas and other biotechnological approaches; Fig. 1) will probably be accepted with few obstacles from regulatory agencies and consumers. Despite the promising initial results reported by Huang et al., and potential to use the host defense peptide to control HLB, it is worth noting that its efficacy at controlling HLB is yet to be verified in field trials, in which adverse environmental factors might reduce its stability and efficacy. Indeed, conducting field trials with large citrus trees, as opposed to young plants in the greenhouse, is the next critical step in testing the feasibility of using MaSAMP and other antimicrobials to control HLB (Fig. 1). In the meantime, it is paramount to further investigate other mechanisms that are responsible for the tolerance/resistance of HLB-tolerant/resistant citrus genotypes (Fig. 1). Synergistic activity of antimicrobial peptides, reactive oxygen species, and other immune responses are expected to work together against Liberibacter infection.