Overview

The ability to detect the presence of pathogens is required for plant cells to respond promptly to invading microorganisms. However, their defense system differs from mammals and invertebrates because plant cells have not developed an acquired immune system to recognize non-self-invaders and generate a specific immune response, i.e., antibodies. Nevertheless, plants can recognize pathogens with two related groups of receptors.¹ The first line of defense, pattern recognition receptors (PRRs), consist of receptors recognizing pathogen/microbe-associated molecular patterns (PAMPs or MAMPs)  or molecules, such as lipopolysaccharides, flagellin, or chitin. These molecules are evolutionarily conserved in microorganisms but not present in plant cells. PRRs are mostly receptor-like proteins/kinases (RLPs or RLKs) attached to plant cell membranes and resemble animal toll-like receptors (TLRs) in structure and function. The resistance resulting from the activity of these receptors is called PAMP-triggered immunity (PTI). If this first defense system fails, plants initiate a second mechanism of defense known as effector-triggered immunity (ETI), of which receptors are mainly intracellular receptors called resistance proteins (R proteins). R proteins detect pathogen effectors entering the host cells to activate the ETI.² Genes encoding R proteins in the ETI have been cloned from various plant species. Many encode the nucleotide-binding site and leucine-rich repeat (NBS-LRR) proteins involved in detecting diverse pathogens, i.e., bacteria, viruses, fungi, nematodes, insects, and oomycetes.

Most commercial grape cultivars are Vitis vinifera species susceptible to grape pathogens, but others, i.e., Vitis amurensis, Vitis riparia, or Vitis rupestris, are tolerant/resistant. The difference in resistance and susceptibility may partly be due to the presence or regulation of R protein (NBS-LRR) expressions in the tolerant/resistant plants. The Toll-interleukin-1 receptor/coiled-coil (TIR/CC) N-terminal, NBS, and LRR are three protein domains of NBS-LRR proteins. Each domain plays a specific role in protein function (Figure 1-1a). Previously, Di Gaspero and Cipriani isolated a set of NBS-containing sequences from the genomic DNA of two resistant grape species with different resistance levels against downy mildew and other pathogens.³ Using the degenerate-PCR primers LM637 and LM638, which correspond to DNA encoding two conserved motifs—GLPL and P-loop—within the NBS domain (Figure 1-1b), they obtained > 12 grape NBS sequences highly similar to those of known R-genes from other plants, which indicates that the cloned sequences belong to the NBS gene family and classify into three major groups. One sequence from each group of probes for Southern blots of resistant and susceptible grapes showed different banding patterns between the two grapes, making them candidate markers for disease-resistance genes in grape germplasm. Thus, NBS sequences or NBS-LRR genes can be used as selection markers in the grape breeding program.

Restriction enzyme-based and polymerase chain reaction (PCR) cloning are two standard methods for molecular cloning. The former uses the same restriction enzyme(s) to cut the cloning vector and the target DNA to be cloned. The digested DNAs are isolated after agarose gel electrophoresis, which is then ligated into the vector. This method relies on the availability of restriction recognition sites in the region flanking the target DNA without cutting inside. Nevertheless, target DNA is not guaranteed to contain the needed restriction sites. Isolating restriction-digested DNA fragments by agarose gel electrophoresis can be difficult because there may be others of the same or similar-sized fragments when cloning DNA from a large genome like grapes. As an alternative, sequences can be added into primers to create flanking restriction sites or other overhangs for PCR cloning. PCR can also produce a large quantity of DNA with a single or minimal number of cloning DNA fragments after simple purification due to the specificity of the primers.

Although PCR cloning carries a higher risk for mutation than restriction enzyme-based cloning, it has become popular because of its simple and quick procedure. Unintentional mutations by PCR can be identified and eliminated by sequencing the PCR amplified DNA. An example of PCR cloning is TOPO TA cloning, in which the non-proofreading Taq DNA polymerase adds adenosine (A) to the 3′-end of the PCR product in a non-template-dependent manner.⁴ This extra A at the 3′ end of each strand of double-stranded PCR-amplified DNA can hybridize to the Thymine (T) overhangs in a linearized cloning vector attached to topoisomerase I to mediate strand ligation (Figure 1-2). For the StrataClone TOPO Cloning Kit used in this lab module, instead of Thymine (T) overhangs, the extra A hybridizes to Uridine (U) overhangs in a linearized cloning vector attached to the topoisomerase I for strand ligation.

This lab module aims to teach you how to practice degenerate PCR and TOPO cloning of nucleotide-binding site (NBS) sequences from grapes. The three weeks’ lab exercises of this module include (i) isolating and purifying genomic DNA, which is then amplified using degenerate primers corresponding to the P-loop and GLPL motifs within the NBS domain (week 1), (ii) validating the size of PCR product(s) by agarose gel electrophoresis; purifying the PCR product or extracting the DNA from the agarose gel if  > 1 band is present, and practicing degenerate PCR primer design (week 2), and (iii) cloning the purified product in a cloning vector and screening for bacterial cells harboring the recombinant plasmid (week 3). The outline of the weekly lab exercise is shown below (Figure 1-3).